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United States Patent |
5,688,774
|
Jacobson
,   et al.
|
November 18, 1997
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A.sub.3 adenosine receptor agonists
Abstract
The present invention provides A.sub.3 selective agonists, particularly,
adenine compounds having selected substituents at the 2, 6, and 9
positions, and related substituted compounds, particularly those
containing substituents on the benzyl and/or uronamide groups, as well as
pharmaceutical compositions containing such compounds. The present
invention also provides a method of selectively activating an A.sub.3
adenosine receptor in a mammal, which method comprises acutely or
chronically administering to a mammal in need of selective activation of
its A.sub.3 adenosine receptor a therapeutically or prophylactically
effective amount of a compound which binds with the A.sub.3 receptor so as
to stimulate an A.sub.3 receptor-dependent response.
Inventors:
|
Jacobson; Kenneth A. (Silver Spring, MD);
Jeong; Heaok Kim (Rockville, MD);
Siddiqi; Suhaib M. (Gaithersburg, MD);
Johnson; Carl R. (Detroit, MI);
Secrist, III; John A. (Birmingham, AL);
Tiwari; Kamal N. (Birmingham, AL)
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Assignee:
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The United States of America as represented by the Department of Health (Washington, DC)
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Appl. No.:
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396111 |
Filed:
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February 28, 1995 |
Current U.S. Class: |
514/46; 514/45; 536/26.7; 536/27.14 |
Intern'l Class: |
A61K 031/70; C07H 019/167; C07H 019/173 |
Field of Search: |
514/45,46
536/26.7,27.14
|
References Cited
Other References
Cermak et al., "(.+-.) 4.beta.-Amino-2.alpha.,
3.alpha.-Dihydroxy-.beta.-Cylcopentanemethanol Hydrochloride. Carbocyclic
Ribofuranosylamine for the Synthesis of Carbocyclic Nucleosides,"
Tetrahedron Lett., 22, 2331-2332 (1981) mos. not available.
Cheng et al., "Relationships between the Inhibition Constant (K.sub.I) and
the Concentration of Inhibitor which causes 50 Per Cent Inhibition
(I.sub.50) of an Enzymatic Reaction," Biochem. Pharmacol., 22, 3099-3108
(1973) mos. not available.
Ji et al., "A Selective Agonist Affinity Label for A.sub.3 Adenosine
Receptors," BioChemical and Biophysical Research Communications, 203,
570-576 (Aug. 30, 1994) mos. not available.
Ji et al., "Species Differences in Ligand Affinity at Central A.sub.3
-Adenosine Receptors," Drug Development Research, 33, 51-59 (1994) mos.
not available.
Johnson et al., "Chemoenzymatic Synthesis of 4-Substituted Riboses.
S(4'-Methyladenosyl)-L-homocysteine," J. Org. Chem., 59, 5854-5855 (1994)
mos. not available.
Kim et al., "Selective Ligands for Rat A.sub.3 Adenosine Receptors:
Structure-Activity Relationships of 1,3-Dialkylxanthine 7-Riboside
Derivatives," J. Med. Chem., 37, 4020-4030 (1994) mos. not available.
Kim et al., "Structure -Activity Relationships of 1,3-Dialkylxanthine
Derivatives at Rat A.sub.3 Adenosine Receptors", Journal of Medicinal
Chemistry, 3373-3382 (Sep. 1994) mos. not available.
Kim et al., "2-Substitution of N.sup.6 -Benzyladenosine-5'-uronamides
Enhances Selectivity for A.sub.3 Adenosine Receptors," Journal of
Medicinal Chemistry, 3614-3621 (Oct. 1994) mos. not available.
Mungall et al., "Use of the Azido Group in the synthesis of 5' Terminal
Aminodeoxythmidine Oligonucleotides," J. Org. Chem., 40, 1659-1662 (1975)
mos. not available.
Olah et al., ".sup.125 I-4-Aminobenzyl-5'-N-methylcarboxamidoadenosine, a
High Affinity Radioligand for the Rat A.sub.3 Adenosine Receptor,"
Molecular Pharmacology, 45, 978-982 (May 1994) mos. not available.
Siddiqi et al., "Quantitative Structure-Activity Studies of Selective A3
Adenosine Agonists," Abstract and Poster Presentation, Amer. Chem. Soc.
Meeting, Washington, D.C. (Aug. 1994) mos. not available.
Siddiqi et al., "Enantiospecific Synthesis of 5'-Noraristeromycin and its
7-Deaza Derivative and a Formal Synthesis of (-)-5'-Homoartisteromycin,"
Nuclesides & Nucleotides, 12, 267-278 (1993) mos. not available.
Stiles et al., "The A.sub.1 Adenosine Receptor: Identification of the
Binding Subunit by Photoaffinity Cross-Linking," J. Biol. Chem., 260,
10806-10811 (1985) mos. not available.
Tiwari et al., "Synthesis and Biological Activity of 4'-Thionucleosides of
2-Chloroadenine," Nucleosides & Nucleotides, 13, 1819-1828 (1994) mos. not
available.
Von Lubitz et al., "Adenosine A.sub.3 receptor stimulation and cerebral
ischemia," European Journal of Pharmacology, 263, 59-67 (1994) mos. not
available.
Von Lubitz et al., "The effects of adenosine A.sub.3 receptor stimulation
on seizures in mice," European Journal of Pharmacology, 275, 23-29 (1995)
mos. not available.
Vorbruggen et al., "Nucleoside Synthesis with Trimethylsilyl triflate and
Perchlorate as Catalysts," Chem. Ber. 114, 1234-1255 (1981) mos. not
available.
|
Primary Examiner: Kunz; Gary L.
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Parent Case Text
This application is a continuation-in-part of copending U.S. patent
application Ser. No. 08/274,628, filed Jul. 13, 1994, which is a
continuation-in-part of U.S. patent application Ser. No. 08/163,324, filed
Dec. 6, 1993, now abandoned, which, in turn, is a continuation-in-part of
U.S. patent application Ser. No. 08/091,109, filed Jul. 13, 1993 and now
abandoned.
Claims
What is claimed is:
1. A compound of the formula:
##STR14##
wherein R.sub.1 is
##STR15##
wherein X.sub.1 is hydrogen or C.sub.1 -C.sub.10 alkyl, X.sub.2 is C.sub.1
-C.sub.10 alkylamido, and each of X.sub.3 and X.sub.4 is independently
hydrogen, hydroxyl, amino, azido, halo, OCOPh,
##STR16##
or both X.sub.3 and X.sub.4 are oxygen connected to >C.dbd.S to form a
5-membered ring, or X.sub.2 and X.sub.3 form the ring
##STR17##
where R' and R" are independently C.sub.1 -C.sub.10 alkyl, with the
proviso that both X.sub.3 and X.sub.4 are not hydroxyl when X.sub.1 is
hydrogen; R.sub.2 is hydrogen, halo, or C.sub.1 -C.sub.10 alkylamino; and
R.sub.3 is benzyl or halobenzyl.
2. The compound of claim 1, wherein said compound is selected from the
group consisting of
2-chloro-9-(2'-amino-2',3'-dideoxy-.beta.-D-5'-methyl-arabino-furonamido)-
N.sup.6 -(3-iodobenzyl)adenine,
2-chloro-9-(2',3'-dideoxy-2'-fluoro-.beta.-D-5'-methyl-arabinofuronamido)-
N.sup.6 -(3-iodobenzyl)adenine,
9-(2-acetyl-3-deoxy-.beta.-D-5-methyl-ribofuronamido)-2-chloro-N.sup.6
(3-iodobenzyl)adenine,
2-chloro-9-(3-deoxy-2-methanesulfonyl-.beta.-D-5-methyl-ribofuronamido)-N.
sup.6 -(3-iodobenzyl)adenine,
2-chloro-9-(3-deoxy-.beta.-D-5-methyl-ribofuronamido)-N.sup.6
-(3-iodobenzyl)adenine,
2-chloro-9-(3,5-1,1,3,3-tetraisopropyldisiloxyl-.beta.-D-5-ribofuranosyl)-
N.sup.6 -(3-iodobenzyl)adenine,
2-chloro-9-(2',3'-O-thiocarbonyl-.beta.-D-5-methyl ribofuronamido)-N.sup.6
-(3-iodobenzyl)adenine,
9-(2-phenoxythiocarbonyl-3-deoxy-.beta.-D-5-methyl-ribofuronamido)
2-chloro-N.sup.6 -(3-iodobenzyl)adenine,
1-(6-benzylamino-9H-purin-9-yl)-1-deoxy-N,4-dimethyl-.beta.-Dribofuranosid
uronamide,
2-chloro-9-(2,3-dideoxy-.beta.-D-5-methyl-ribofuronamido)-N.sup.6
-benzyladenine,
2-chloro-9-(2'-azido-2',3'-dideoxy-.beta.-D-5'-methyl-arabinofuronamido)-N
.sup.6 -benzyladenine, and 2-chloro-9-(.beta.-D-erythrofuranoside)-N.sup.6
-(3-iodobenzyl) adenine.
3. A compound of the formula:
##STR18##
wherein R.sub.1 is
##STR19##
wherein X.sub.1 is hydrogen, X.sub.2 is hydrogen or C.sub.1 -C.sub.10
alkylamido, X.sub.3 and X.sub.4 are hydrogen or hydroxyl, R.sub.2 is
hydrogen, halo, or C.sub.1 -C.sub.10 alkylamino, and R.sub.3 is
benzodioxanemethyl, furfuryl, L-prolylaminobenzyl,
.beta.-alanylaminobenzyl, T-BOC-.beta.-alanylaminobenzyl, phenylamino, or
phenoxy.
4. The compound of claim 3, wherein said compound is selected from the
group consisting of N.sup.6 -(benzodioxanemethyl)adenosine,
1-(6-furfurylamino-9H-purin-9-yl)-1-deoxy-N-methyl-.beta.-D-ribofuranosidu
ronamide, N.sup.6 -›3-(L-prolylamino)benzyl!adenosine-5'-N-methyluronamide,
N.sup.6 -›3-(.beta.-alanylamino) benzyl!adenosine-5'-N-methyluronamide,
N.sup.6
-›3-(N-T-Boc-.beta.-alanylamino)benzyl!adenosine-5'-N-methyluronamide,
6-(N'-phenylhydrazinyl)purine-9-.beta.-ribofuranoside-5'-N-methyluronamide
, and
6-(O-phenylhydroxylamino)purine-9-.beta.-ribofuranoside-5'-N-methyluronami
de.
5. A compound of the formula:
##STR20##
wherein R.sub.1 is
##STR21##
wherein X.sub.1 is hydrogen, and X.sub.2 is C.sub.1 -C.sub.10
hydroxyalkyl, R.sub.2 is halo or C.sub.1 -C.sub.10 alkylamino, and R.sub.3
is halobenzyl.
6. The compound of claim 5, wherein said compound is
9-(.beta.-D-erythrofuranoside)-2-methylamino-N.sup.6
-(3-iodobenzyl)adenine or
2-chloro-N-(3-iodobenzyl)-9-(2-tetrahydrofuryl)-9H-purin-6-amine.
7. A compound of the formula
##STR22##
wherein R.sub.1 is
##STR23##
wherein X.sub.1 is hydrogen or C.sub.1 -C.sub.10 hydroxyalkyl, X.sub.2
and X.sub.3 are independently hydrogen, hydroxyl, or halo, R.sub.2 is
hydrogen or halo, and R.sub.3 is hydrogen, benzyl, or halobenzyl.
8. The compound of claim 7, wherein said compound is
2-chloro-(2'-deoxy-6'-thio-L-arabinosyl)adenine or
2-chloro-(6'-thio-L-arabinosyl)adenine.
9. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier and a therapeutically effective amount of the compound of claim 1.
10. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier and a therapeutically effective amount of the compound of claim 2.
11. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier and a therapeutically effective amount of the compound of claim 5.
12. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier and a therapeutically effective amount of the compound of claim 7.
13. A method of selectively activating an A.sub.3 adenosine receptor in a
mammal, which method comprises administering to a mammal in need of
selective activation of its A.sub.3 adenosine receptor a therapeutically
effective amount of a compound of claim 1.
14. A method of selectively activating an A.sub.3 adenosine receptor in a
mammal, which method comprises administering to a mammal in need of
selective activation of its A.sub.3 adenosine receptor a therapeutically
effective amount of a compound of claim 3.
15. A method of selectively activating an A.sub.3 adenosine receptor in a
mammal, which method comprises administering to a mammal in need of
selective activation of its A.sub.3 adenosine receptor a therapeutically
effective amount of a compound of claim 5.
16. A method of selectively activating an A.sub.3 adenosine receptor in a
mammal, which method comprises administering to a mammal in need of
selective activation of its A.sub.3 adenosine receptor a therapeutically
effective amount of a compound of claim 7.
Description
TECHNICAL FIELD OF THE INVENTION
The present invention relates to A.sub.3 adenosine receptor agonists and
methods of selectively activating an A.sub.3 adenosine receptor in a
mammal. The present invention also relates to methods of treating various
medical disorders with A.sub.3 adenosine receptor agonists.
BACKGROUND OF THE INVENTION
Adenosine receptors, belonging to the superfamily of the G protein-coupled
receptors, are generally divided into two major subclasses, A.sub.1 and
A.sub.2, on the basis of the differential affinities of a number of
adenosine receptor agonists and antagonists for the receptors, their
primary structures, and the secondary messenger systems to which they
couple. Thus, A.sub.2 receptors, which can be further subdivided into
A.sub.2a and A.sub.2b, stimulate adenylate cyclase, whereas A.sub.1
receptors may couple to a variety of secondary messenger systems,
including those involved in the inhibition of adenylate cyclase, the
inhibition or stimulation of phosphoinositol turnover, the activation of
guanylate cyclase, the activation of potassium influx, and the inhibition
of calcium influx (van Galen et al., Med. Res. Rev., 12, 423-471 (1992);
Jacobson et al., J. Med. Chem., 35, 407-422 (1992)).
Recently, a novel adenosine receptor was identified on the basis of its
primary structure and cloned from rat brain (Zhou et al., Proc. Natl.
Acad. Sci. U.S.A., 89, 7432-7436 (1992)) and rat testis (Meyerhof et al.,
FEBS Lett., 284, 155-160 (1991)). The putative transmembrane domains of
the novel adenosine receptor, which has been designated the A.sub.3
receptor, show 58% identity with the canine A.sub.1 receptor and 57% with
the canine A.sub.2a receptor. Like the A.sub.1 receptor, the A.sub.3
receptor is negatively coupled to adenylate cyclase (Zhou et al.).
The potential utility of A.sub.1 - and A.sub.2 -selective agents in
therapeutic applications has been limited by accompanying side effects,
given the ubiquitous nature of the A.sub.1 and A.sub.2 receptors. The
distribution of the A.sub.3 receptor, by contrast, is fairly limited,
being found primarily in the central nervous system (CNS) (Zhou et al.),
brain, testes (Meyerhof et al.), and immune system, where it appears to be
involved in the modulation of release from mast cells of mediators of the
immediate hypersensitivity reaction (Ramkumar et al., J. Biol. Chem., 268,
16887-16890 (1993)). The limited distribution of the A.sub.3 receptor
provides a basis for predicting that A.sub.3 -selective compounds may be
more useful than A.sub.1 - and A.sub.2 -selective compounds as potential
therapeutic agents. It is believed that A.sub.3 -selective compounds will
have utility in the therapeutic and/or prophylactic treatment of cardiac
disease, infertility, kidney disease, and CNS disorders.
Few ligands for this novel receptor have been reported. Some non-selective
N.sup.6 -substituted adenosine derivatives have been described as agonists
for the A.sub.3 receptor, including APNEA (N.sup.6
-2-(4-aminophenyl)ethyladenosine), which has been used successfully as a
radioligand in its iodinated form (Zhou et al.). Typical xanthine and
nonxanthine A.sub.1 and A.sub.2 receptor antagonists, however, do not
appear to bind to this receptor (Zhou et al.).
Thus, there remains a need for A.sub.3 -selective agonists. The present
invention seeks to provide such compounds, as well as methods of using
these compounds to selectively activate the A.sub.3 receptor in mammals,
and pharmaceutical compositions comprising such compounds. These and other
objects and advantages of the present invention, as well as additional
inventive features, will be apparent from the description of the invention
provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides A.sub.3 selective agonists, particularly,
adenine compounds having selected substituents at the 2, 6, and 9
positions, and related substituted compounds, particularly those
containing substituents on the benzyl and/or uronamide groups, as well as
pharmaceutical compositions containing such compounds. The present
invention also provides a method of selectively activating an A.sub.3
adenosine receptor in a mammal, which method comprises acutely or
chronically administering to a mammal in need of selective activation of
its A.sub.3 adenosine receptor a therapeutically or prophylactically
effective amount of a compound which binds with the A.sub.3 receptor so as
to stimulate an A.sub.3 receptor-dependent response.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram depicting the chemical synthesis of 9-methyl
substituted adenine derivatives 3, 6, 9, and 10-17.
FIG. 2 is a schematic diagram depicting the chemical synthesis of adenine
derivatives 18-22 having hydroxyalkyl, carboxyalkyl, and cyanoalkyl
substituents at the 9-position, and the synthesis of adenine compounds
23-24 having selected substituents at the 2-position.
FIG. 3 is a schematic diagram depicting the chemical synthesis of the
9-erythrose derivative 28.
FIG. 4 is a schematic diagram depicting the chemical synthesis of modified
ribose analogues.
FIG. 5 is a schematic diagram depicting the chemical synthesis of the
2',3'-dideoxy adenosine compound 40.
FIG. 6 is a schematic diagram depicting the chemical conversion of compound
38 to compounds 41-44, having methanesulfonyl, azido, amino, and fluoro
substituents.
FIG. 7 is a schematic diagram depicting the chemical synthesis of the
carbocyclic derivative 51.
FIG. 8 is a schematic diagram depicting the chemical transformation of the
methyl uronate 52 into the corresponding nucleoside.
FIG. 9 is a schematic diagram depicting the chemical synthesis of the
carbocyclic derivatives (.+-.) 61.
FIG. 10 is a schematic diagram depicting the chemical synthesis of
compounds 62-67.
FIG. 11 is a schematic diagram depicting the chemical synthesis of
compounds 68-71.
FIG. 12 is a graph depicting the agonist-elicited inhibition of adenylate
cyclase via rat A3 receptors in transfected CHO cells wherein % adenylate
cyclase activity is plotted against concentration (-log M): circles, NECA;
squares, 2-Cl-IB-MECA; triangles, compound 41b.
FIG. 13 is a graph depicting the agonist-elicited inhibition of adenylate
cyclase via rat A3 receptors in transfected CHO cells wherein % adenylate
cyclase activity is plotted against concentration (-log M): circles,
2-Cl-IB-MECA; squares, NECA; triangles,
N6-benzyl-4'methyladenosine-5'-N-methyluronamide.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides compounds which have been found to be
selective A.sub.3 adenosine receptor agonists, pharmaceutical compositions
containing such compounds, and related treatment methods and assay
methods.
The modification of adenosine at the 5'-position and/or at the N.sup.6
-position with groups that enhance A.sub.3 potency has been found to
result in moderate A.sub.3 selectivity. In particular, the
5'-methyluronamide modification of adenosine and the N.sup.6 -benzyl
group, either alone or in combination, increases affinity in binding to
A.sub.3 receptors relative to A.sub.1 and A.sub.2a receptors. Optimization
of substituent groups has led to the development of the highly potent
A.sub.3 agonist N.sup.6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide
(IB-MECA) which is 50-fold selective for A.sub.3 vs. either A.sub.1 or
A.sub.2 receptors. A closely related, but less selective radioligand,
›.sup.125 I!AB-MECA, has been developed for characterization of A.sub.3
receptors and has been found to have a K.sub.d value of 3.6 nM in binding
to rat A.sub.3 receptors in the RBL-2H3 mast cell line. While derivatives
such as N.sup.6 -benzyladenosine-5'-N-ethyluronamide have been found to be
full agonists in inhibiting adenylate cyclase via rat A.sub.3 receptors,
such derivatives, while useful, are only one order of magnitude selective
for rat A.sub.3 receptors vs. either A.sub.1 or A.sub.2a receptors in
binding assays.
Triple substitution of adenosine results in the further enhancement of the
degree of A.sub.3 selectivity, such that an improvement in selectivity in
binding assays of three orders of magnitude or more can be achieved. By
combining the two modifications at 5'- and N.sup.6 -positions, which
result in moderate selectivity, with a third site of modification,
particularly the 2-position, selectivity can be dramatically increased.
For example, 2-chloro-N.sup.6
-(3-iodobenzyl)-adenosine-5'-N-methyluronamide has been found to be the
most potent and selective agent in binding assays and has been shown to be
a full agonist in the inhibition of adenylate cyclase. The agonist potency
was also greater than that of other agonists, indicating a parallel
between binding affinities and relative potencies in this functional
assay. Such agonist properties are similarly expected in another relevant
functional assay, namely stimulation of A.sub.3 -mediated phosphoinositide
metabolism.
The novel A.sub.3 adenosine receptor is believed to be important in the
regulation of CNS, cardiac, inflammatory, and reproductive functions.
Activation of A.sub.3 receptors enhances the release of inflammatory
mediators from mast cells (Ramkumar et al., J. Biol. Chem., 268,
16887-16890 (1993); Ali et al., J. Biol. Chem., 265, 745-753 (1990)),
lowers blood pressure (Fozard et al., Br. J. Pharmacol., 109, 3-5 (1993)),
and depresses locomotor activity (Jacobson et al., FEBS Letters, 336,
57-60 (1993)). Selective agonists are believed to have therapeutic
potential as cerebroprotective agents (von Lubitz et al., Drug Devel.
Res., 31, 332 (Abstract 1224) (1994), and the activation of A.sub.3
receptors is thought to be related to the cardioprotective preconditioning
response following exposure to adenosine agonists. It has been discovered
that the chronic administration of an A.sub.3 agonist provides a
cerebroprotective effect. For example, the cerebroprotective effects of
IB-MECA have been discovered using an ischemic model in gerbils and
NMDA-induced seizures in mice (von Lubitz et al., Neurosci Abstr. (1994)).
Moreover, the cardioprotective potential of A.sub.3 receptor activation,
based on use of APNEA coadministered with a xanthine antagonist that does
not act at A.sub.3 receptors, has been demonstrated. APNEA has been found
to be 8-fold A.sub.1 selective, and its pharmacological use is limited to
such combination with antagonists of both A.sub.1 and A.sub.2a receptors.
Clearly, the availability of ligands such as those described herein,
particularly 2-chloro-N.sup.6
-(3-iodobenzyl)-9-›5-(methylamido)-.beta.-D-ribofuranosyl!-adenine, could
be critical in pharmacological studies of A.sub.3 receptors. A highly
selective A.sub.3 ligand is expected to be especially useful as a
radioligand, since the currently used high affinity ligand ›.sup.125
I!AB-MECA, is not sufficiently selective for general application in tissue
(Olah et al., Mol. Pharmacol., 45, 978-982 (1994)).
Although the selectivities of these novel A.sub.3 agonists may vary
somewhat in different species, due to the unusually large species
dependence in ligand affinity at this subtype, such differences appear to
be more pronounced for antagonists than for agonists (Linden et al., Mol.
Pharmacol., 44, 524-532 (1993); Salvatore et al., Proc. Natl. Acad. Sci.
U.S.A., 90, 10365-10369 (1993); Brackett et al., Biochem. Pharmacol., 47,
801-814 (1994)). Thus, it should be noted that 2-chloroadenosine is
17-fold less potent than NECA at rat A.sub.3 receptors, whereas at sheep
A.sub.3 receptors 2-chloroadenosine is 1.7-fold less potent than NECA.
Thus, since the most selective compound in the present series,
2-chloro-N.sup.6
-(3-iodobenzyl)-9-›5-(methylamido)-.beta.-D-ribofuranosyl!-adenine,
contains the 2-chloro substitution, it is likely that the selectivity will
not be substantially diminished in other species, such as sheep and human.
A high degree of correlation in the relative affinities of adenosine
derivatives at rat vs. human A.sub.3 receptors has been shown.
A high degree of selectivity exists for doubly-substituted derivatives,
such as N.sup.6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA),
and triply-substituted adenosine derivatives for A.sub.3 receptors vs. the
NBTI-sensitive adenosine uptake site. 2-substitution is well-tolerated at
A.sub.3 receptors, whether it be with a small group (e.g.,
2-chloro-N.sup.6 -(3-iodobenzyl)-adenosine) or a large group (e.g., APEC).
The potency enhancing effects of 2-substituents appeared to follow the
order: chloro>thioether>amine. The effects of 2-substitution to enhance
A.sub.3 affinity are also additive with effects of uronamides at the
5'-position and a 3-iodobenzyl group at the N.sup.6 -position, although
the A.sub.3 affinity-enhancing effect of a 2-chloro group do not appear to
be additive with an N.sup.6 -cyclopentyl group. The combination of most
favorable modifications at three positions has led to very potent and
highly selective agonist ligands.
Compounds
The present invention provides a compound having the formula
##STR1##
wherein
R.sub.1 is C.sub.1 -C.sub.10 alkyl, C.sub.1 -C.sub.10 hydroxyalkyl, C.sub.1
-C.sub.10 carboxyalkyl, or C.sub.1 -C.sub.10 cyanoalkyl, R.sub.2 is
hydrogen, halo, amino, hydrazido, C.sub.1 -C.sub.10 alkylamino, C.sub.1
-C.sub.10 alkoxy, C.sub.1 -C.sub.10 thioalkoxy, or pyridylthio, and
R.sub.3 is benzyl or halobenzyl. A preferred compound of the above formula
is a compound having iodobenzyl as R.sub.3. Examples of preferred
compounds include .sup.6 -(3-iodobenzyl)-9-methyladenine, N.sup.6
-(3-iodobenzyl)-9-hydroxyethyladenine, R--N.sup.6
-(3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine, S--N.sup.6
-(3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine, N.sup.6
-(3-iodobenzyladenin-9-yl)acetic acid, N.sup.6
-(3-iodobenzyl)-9-(3-cyanopropyl)adenine, 2-chloro-N.sup.6
-(3-iodobenzyl)-9-methyladenine, 2-amino-N.sup.6
-(3-iodobenzyl)-9-methyladenine, 2-hydrazido-N.sup.6
-(3-iodobenzyl)-9-methyladenine, N.sup.6
-(3-iodobenzyl)-2-methylamino-9-methyladenine, 2-dimethylamino-N.sup.6
-(3-iodobenzyl)-9-methyladenine, N.sup.6
-(3-iodobenzyl)-9-methyl-2-propylaminoadenine, 2-hexylamino-N.sup.6-(
3-iodobenzyl)-9-methyladenine,N.sup.6
-(3-iodobenzyl)-2-methoxy-9-methyladenine, N.sup.6
-(3-iodobenzyl)-9-methyl-2-methylthioadenine, and N.sup.6
-(3-iodobenzyl)-9-methyl-2-(4-pyridylthio)adenine.
The present invention also provides a compound of the formula
##STR2##
wherein R.sub.1 is
##STR3##
wherein X.sub.1 is hydrogen or C.sub.1 -C.sub.10 alkyl, and X.sub.2 is
hydroxyl or C.sub.1 -C.sub.10 alkylamido, R.sub.2 is halo, amino, or
phenyl C.sub.1 -C.sub.10 alkylamino, and R.sub.3 is hydrogen, benzyl, or
halobenzyl. Examples of preferred compounds include
(1S,2R,3S,4R)-4-(6-amino-2-phenylethylamino-9H-purin-9-yl)cyclopentane-1,2
,3-triol, (1S,2R,3S,4R)-4-(6-amino-2-chloro-9H-purin-9-yl)
cyclopentane-1,2,3-triol, and
(.+-.)-9-›2.alpha.,3.alpha.-dihydroxy-4.beta.-(N-methylcarbamoyl)cyclopent
-1.beta.-yl)!-N.sup.6 -(3-iodobenzyl)-adenine.
The present invention further provides a compound of the formula:
##STR4##
wherein R.sub.1 is
##STR5##
wherein X.sub.1 is hydrogen or C.sub.1 -C.sub.10 alkyl, X.sub.2 is C.sub.1
-C.sub.10 alkylamido, and each of X.sub.3 and X.sub.4 is independently
hydrogen, hydroxyl, amino, azido, halo, OCOPh,
##STR6##
or both X.sub.3 and X.sub.4 are oxygen connected to >C.dbd.S to form a
5-membered ring, or X.sub.2 and X.sub.3 form the ring
##STR7##
where R' and R" are independently C.sub.1 -C.sub.10 alkyl, with the
proviso that both X.sub.3 and X.sub.4 are not hydroxyl when X.sub.1 is
hydrogen, R.sub.2 is hydrogen, halo, or C.sub.1 -C.sub.10 alkylamino, and
R.sub.3 is benzyl or halobenzyl. Examples of preferred compounds include
2-chloro-9-(2'-amino-2',3'-dideoxy-.beta.-D-5'-methyl-arabino-furonamido)-N
.sup.6 -(3-iodobenzyl)adenine,
2-chloro-9-(2',3'-dideoxy-2'-fluoro-.beta.-D-5'-methyl-arabino
furonamido)-N.sup.6 -(3-iodobenzyl)adenine,
9-(2-acetyl-3-deoxy-.beta.-D-5-methyl-ribofuronamido)-2-chloro-N.sup.6
(3-iodobenzyl)adenine,
2-chloro-9-(3-deoxy-2-methanesulfonyl-.beta.-D-5-methyl-ribofuronamido)-N.
sup.6 -(3-iodobenzyl)adenine,
2-chloro-9-(3-deoxy-.beta.-D-5-methyl-ribofuronamido)-N.sup.6
-(3-iodobenzyl)adenine,
2-chloro-9-(3,5-1,1,3,3-tetraisopropyldisiloxyl-.beta.-D-5-ribofuranosyl)-N
.sup.6 -(3-iodobenzyl)adenine,
2-chloro-9-(2',3'-O-thiocarbonyl-.beta.-D-5-methyl-ribofuronamido)-N.sup.6
-(3-iodobenzyl)adenine,
9-(2-phenoxythiocarbonyl-3-deoxy-.beta.-D-5-methyl-ribofuronamido)-2-chloro
-N.sup.6 -(3-iodobenzyl)adenine,
1-(6-benzylamino-9H-purin-9-yl)-1-deoxy-N,4-dimethyl-.beta.-D-ribofuranosid
uronamide,
2-chloro-9-(2,3-dideoxy-.beta.-D-5-methyl-ribofuronamido)-N.sup.6
benzyladenine,
2-chloro-9-(2'-azido-2',3'-dideoxy-.beta.-D-5'-methyl-arabino-furonamido)-
N.sup.6 -benzyladenine, and 2-chloro-9-(.beta.-D-erythrofuranoside)-N.sup.6
-(3-iodobenzyl)adenine.
The present invention provides, in addition, a compound of the formula:
##STR8##
wherein
R.sub.1 is
##STR9##
wherein X.sub.1 is hydrogen, X.sub.2 is hydrogen or C.sub.1 -C.sub.10
alkylamido, and X.sub.3 and X.sub.4 are hydrogen or hydroxyl, R.sub.2 is
hydrogen, halo, or C.sub.1 -C.sub.10 alkylamino, and R.sub.3 is
benzodioxanemethyl, furfuryl, L-prolylaminobenzyl,
.beta.-alanylaminobenzyl, T-BOC-.beta.-alanylaminobenzyl, phenylamino, or
phenoxy. Examples of preferred compounds include N.sup.6
-(benzodioxanemethyl) adenosine,
1-(6-furfurylamino-9H-purin-9-yl)-1-deoxy-N-methyl-.beta.-D-ribofuranosidu
ronamide, N.sup.6 -›3-(L-prolylamino)benzyl!adenosine-5'-N-methyluronamide,
N.sup.6 -›3-(.beta.-alanylamino) benzyl!adenosine-5'-N-methyluronamide,
N.sup.6
-›3-(N-T-Boc-.beta.-alanylamino)benzyl!adenosine-5'-N-methyluronamide,6-(N
'-phenylhydrazinyl)purine-9-.beta.-ribofuranoside-5'-N-methyluronamide,
6-(O-phenylhydroxylamino)purine-9-.beta.-ribofuranoside-5'-N-methyluronami
de, and 9-(.beta.-D-2',3'-dideoxyerythrofuranosyl)-N.sup.6
-(3-.beta.-alanylamino)benzyl!adenosine.
The present invention also provides the compound having the formula:
##STR10##
wherein
R.sub.1 is
##STR11##
wherein X.sub.1 is hydrogen, and X.sub.2 is C.sub.1 -C.sub.10
hydroxyalkyl, R.sub.2 is halo or C.sub.1 -C.sub.10 alkylamino, and R.sub.3
is halobenzyl. Examples of preferred compounds include
9-(.beta.-D-erythrofuranoside)-2-methylamino-N.sup.6 -(3-iodobenzyl)adenine
and 2-chloro-N-(3-iodobenzyl)-9-(2-tetrahydrofuryl)-9H-purin-6amine.
The present invention also provides a compound of the formula:
##STR12##
wherein
R.sub.1 is
##STR13##
wherein X.sub.1 is hydrogen or C.sub.1 -C.sub.10 hydroxyalkyl,X.sub.2 and
X.sub.3 are independently hydrogen, hydroxyl, or halo, R.sub.2 is hydrogen
or halo, and R.sub.3 is hydrogen, benzyl, or halobenzyl. In accordance
with the instant invention, examples of preferred compounds are
2-chloro-(2'-deoxy-6'-thio-L-arabinosyl)adenine and
2-chloro-(6'-thio-L-arabinosyl)adenine.
All of the aforesaid compounds of the present invention can be used as is
or in the form of a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and a therapeutically effective amount
of the compound.
The present invention also provides a method of selectively activating an
A.sub.3 adenosine receptor in a mammal, which method comprises
administering to a mammal in need of selective activation of its A.sub.3
adenosine receptor a therapeutically effective amount of one or more of
the aforesaid present inventive compounds.
The present invention further provides an assay which comprises providing
one of aforesaid present inventive compounds which has been labeled,
contacting a sample with the labeled compound under conditions sufficient
to effect binding between said labeled compound and a component of the
sample, and determining whether such binding occurred.
Compound Synthesis
The compounds of the present invention, including those useful in the
present inventive compositions and methods, can be synthesized by any
suitable means. FIG. 1 outlines the synthesis of 9-methyl substituted
adenine derivatives.
The synthesis of the 2-unsubstituted adenine derivatives can be carried out
by substitution of the chlorine in compound 1, using 3-iodobenzylamine, to
provide N.sup.6 -(3-iodobenzyl)adenine, 2. This can be followed by
alkylation at the 9-position, resulting in the 9-methyl adenine compound,
3. Alternately, 2-substitution can be introduced at the first synthetic
stage with 2,6-dichloropurine, 4, or with 2-amino-6-chloropurine, 8,
carried through the same sequence, leading to compounds 6 or 9,
respectively. The 2-chloro group can be readily replaced at elevated
temperature by various nucleophiles, such as amines (leading to compounds
10 through 14) or alkoxide or thioalkoxide (leading to compounds 15 and
16). Compound 17 can be produced by the reaction of 6 with sodium
hydrosulfide and pyridine.
FIG. 2 outlines the synthesis of adenine derivatives (18-22) having
hydroxyalkyl, carboxyalkyl, and cyanoalkyl substituents at the 9-position,
and the synthesis of adenine compounds (23-24) substituents at the
2-position. The synthesis of a 9-erythrose derivative, 28, is shown in
FIG. 3.
Synthesis of ribose-modified analogues can start from
5-O-benzoyl-1,2-O-isopropylidene-a-D-xylofuranoside (FIG. 4). The
3-hydroxyl group of the starting material can be deoxygenated by first
forming the xanthate derivative and then reacting the xanthate with
tributyltin hydride and triethylborane to give compound 31. Debenzoylation
of the 5-position and oxidation of resulting alcohol 32 can provide the
acid 33 in good yields. The methylamide at 5-position of compound 35 can
be introduced by esterification of acid to compound 33 and displacement of
the ester group with methylamine in a sealed container. The
1,2-isopropylidene group of compound 35 can be cleaved, and the diol can
be acetylated in a single container by conventional methods to give
compound 38. This sugar intermediate can be condensed as shown in FIG. 5
with a silylated base such as compound 36a by a modified Vorbruggen method
(Chem. Ber., 114, 1234-1255 (1981)) to produce compound 37, the acetyl
group from which can be removed by methanolic ammonia to yield
3'-deoxy-2-chloro-IBMECA, 38. Deoxygenation of compound 39 can produce the
deiodinated 2',3'-dideoxy compound, 40. FIG. 6 depicts the conversion of
compound 38 to compounds 41-44. The .beta.-2'-azide of 42 can be
introduced by displacement of the mesylate group of 41 with sodium azide.
Further, the 2'-azide of compound 42 can be reduced using
triphenylphosphine/ammonium hydroxide in THF-methanol (Mungall et al., J.
Org. Chem., 40, 1659-1662 (1975)) to give the .beta.-2'-amino derivative,
43. The .beta.-2'-fluoro compound, 44, can be synthesized by reaction of
compound 38 with DAST (diethylaminosulfur trifluoride).
A route to the synthesis of the carbocyclic derivative, 51, is shown in
FIG. 7. Using a procedure that has been employed to couple cyclopentyl
acetates with nucleophiles in the presence of Pd(0) catalysis to give cis
oriented products (Siddiqui et al. Nucleosides and Nucleotides, 12,
267-278 (1993)), compound (.+-.)47 can be reacted with 2,6-dichloropurine
to give compound (.+-.)48. Standard vicinal glycolization of compound
(.+-.)48 to compound (.+-.)49 can be followed by exchanging the 6-chloro
group with NH.sub.4 OH to give compound 50. Heating compound 50 with
phenylethylamine will give compound 51.
The methyl 4-methyl-D-ribofuranoside uronate 52 (FIG. 8) can be prepared as
an intermediate in the chemoenzymatic synthesis of methyl
4-methyl-D-ribofuranoside from cyclopentadiene (Johnson et al. J. Org.
Chem., 59, 5854-5855 (1994)). Direct amidation can be carried out with
methylamine in MeOH to give the methyl amide 53, which can be converted to
the diacetate 54 by treatment with HCl/MeOH and acetylation with acetic
anhydride and pyridine. The .beta.-methyl glycoside 54 can be subjected to
Vorbruggen conditions (N.sup.9 -TMS-6-chloropurine, TMS-OTf, CH.sub.3 CN,
50.degree. C.), and the nucleoside initially formed can be converted to,
upon heating to 80.degree. C. for 6-12 h, the thermodynamically more
stable .beta.-nucleoside 55 in good yield. The displacement of the chloro
group with benzylamine can be conducted in t-butylalcohol at 70.degree.
C., to obtain the N-benzyladenosine uronamide, 56.
The synthesis of the carbocyclic analogue (.+-.)61 (FIG. 9) can be
accomplished by heating compound (.+-.)57 with methylamine to provide
compound (.+-.)58. Reaction of compound 58 with 5-amino-4,6-dichloropurine
will give intermediate 59. Heating compound 59 with dimethoxy
methylacetate followed by acidic treatment will give compound 60. The
6-chloro group of compound 60 can be displaced by 3-iodobenzylamine to
give compound 61.
The synthesis of compounds 63-71 is illustrated in FIGS. 10-11. The
2',3'-isopropylidene-6-chloropurine-5'-methyluronamide 61 (Gallo-Rodriguez
et al., J. Med. Chem., 37, 636-646 (1994)) can be heated with
3-aminobenzylamaine or phenylhydrazine to yield compounds 62 and 69,
respectively. Treatment of compound 62 with t-Boc-L-proline or
t-Boc-.beta.-alanine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDAC) in the presence of 4-(N,N-dimethylamino)pyridine and
imidazole will give compounds 63 and 64, respectively. After acidic
treatment, the amino compounds, 65 and 66 can be obtained. Compound 65 or
66 can be treated with di-ter-butyldicarbonate to give compound 67.
Compounds 70 and 71 may be obtained from compounds 69 and 68,
respectively.
The thionucleosides of 2-chloroadenosine such as the thioarabinosides, can
be synthesized following the procedures described in Tiwari et al.
Nucleosides & Nucleotides, 13, 1819-1828 (1994).
Pharmaceutical Compositions
The present invention also provides a pharmaceutical composition comprising
a pharmaceutically acceptable carrier and an effective amount, e.g., a
therapeutically effective amount, including a prophylactically effective
amount, of one or more of the aforesaid compounds of the present
invention.
The carrier can be any of those conventionally used and is limited only by
chemico-physical considerations, such as solubility and lack of reactivity
with the compound, and by the route of administration. It will be
appreciated by one of skill in the art that, in addition to the following
described pharmaceutical compositions, the compounds of the present
invention can be formulated as inclusion complexes, such as cyclodextrin
inclusion complexes, or liposomes.
Examples of pharmaceutically acceptable acid addition salts for use in the
present inventive pharmaceutical composition include those derived from
mineral acids, such as hydrochloric, hydrobromic, phosphoric,
metaphosphoric, nitric and sulphuric acids, and organic acids, such as
tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,
gluconic, succinic, and arylsulphonic, for example p-toluenesulphonic,
acids.
The pharmaceutically acceptable carriers described herein, for example,
vehicles, adjuvants, excipients, or diluents, are well-known to those who
are skilled in the art and are readily available to the public. It is
preferred that the pharmaceutically acceptable carrier be one which is
chemically inert to the active compounds and one which has no detrimental
side effects or toxicity under the conditions of use.
The choice of carrier will be determined in part by the particular active
agent, as well as by the particular method used to administer the
composition. Accordingly, there is a wide variety of suitable formulations
of the pharmaceutical composition of the present invention. The following
formulations for oral, aerosol, parenteral, subcutaneous, intravenous,
intraarterial, intramuscular, interperitoneal, intrathecal, rectal, and
vaginal administration are merely exemplary and are in no way limiting.
Formulations suitable for oral administration can consist of (a) liquid
solutions, such as an effective amount of the compound dissolved in
diluents, such as water, saline, or orange juice; (b) capsules, sachets,
tablets, lozenges, and troches, each containing a predetermined amount of
the active ingredient, as solids or granules; (c) powders; (d) suspensions
in an appropriate liquid; and (e) suitable emulsions. Liquid formulations
may include diluents, such as water and alcohols, for example, ethanol,
benzyl alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant, suspending agent, or
emulsifying agent. Capsule forms can be of the ordinary hard- or
soft-shelled gelatin type containing, for example, surfactants,
lubricants, and inert fillers, such as lactose, sucrose, calcium
phosphate, and corn starch. Tablet forms can include one or more of
lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,
microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon
dioxide, croscarmellose sodium, talc, magnesium stearate, calcium
stearate, zinc stearate, stearic acid, and other excipients, colorants,
diluents, buffering agents, disintegrating agents, moistening agents,
preservatives, flavoring agents, and pharmacologically compatible
carriers. Lozenge forms can comprise the active ingredient in a flavor,
usually sucrose and acacia or tragacanth, as well as pastilles comprising
the active ingredient in an inert base, such as gelatin and glycerin, or
sucrose and acacia, emulsions, gels, and the like containing, in addition
to the active ingredient, such carriers as are known in the art.
The compounds of the present invention, alone or in combination with other
suitable components, can be made into aerosol formulations to be
administered via inhalation. These aerosol formulations can be placed into
pressurized acceptable propellants, such as dichlorodifluoromethane,
propane, nitrogen, and the like. They also may be formulated as
pharmaceuticals for non-pressured preparations, such as in a nebulizer or
an atomizer.
Formulations suitable for parenteral administration include aqueous and
non-aqueous, isotonic sterile injection solutions, which can contain
anti-oxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and aqueous
and non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. The
compound can be administered in a physiologically acceptable diluent in a
pharmaceutical carrier, such as a sterile liquid or mixture of liquids,
including water, saline, aqueous dextrose and related sugar solutions, an
alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such
as propylene glycol or polyethylene glycol, glycerol ketals, such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as
poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or
glyceride, or an acetylated fatty acid glyceride with or without the
addition of a pharmaceutically acceptable surfactant, such as a soap or a
detergent, suspending agent, such as pectin, carbomers, methylcellulose,
hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying
agents and other pharmaceutical adjuvants.
Oils, which can be used in parenteral formulations include petroleum,
animal, vegetable, or synthetic oils. Specific examples of oils include
peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral.
Suitable fatty acids for use in parenteral formulations include oleic
acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl
myristate are examples of suitable fatty acid esters. Suitable soaps for
use in parenteral formulations include fatty alkali metal, ammonium, and
triethanolamine salts, and suitable detergents include (a) cationic
detergents such as, for example, dimethyl dialkyl ammonium halides, and
alkyl pyridinium halides, (b) anionic detergents such as, for example,
alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and
monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such
as, for example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such
as, for example, alkyl-.beta.-aminopropionates, and 2-alkyl-imidazoline
quaternary ammonium salts, and (3) mixtures thereof.
The parenteral formulations will typically contain from about 0.5 to about
25% by weight of the active ingredient in solution. Suitable preservatives
and buffers can be used in such formulations. In order to minimize or
eliminate irritation at the site of injection, such compositions may
contain one or more nonionic surfactants having a hydrophile-lipophile
balance (HLB) of from about 12 to about 17. The quantity of surfactant in
such formulations ranges from about 5 to about 15% by weight. Suitable
surfactants include polyethylene sorbitan fatty acid esters, such as
sorbitan monooleate and the high molecular weight adducts of ethylene
oxide with a hydrophobic base, formed by the condensation of propylene
oxide with propylene glycol. The parenteral formulations can be presented
in unit-dose or multi-dose sealed containers, such as ampules and vials,
and can be stored in a freeze-dried (lyophilized) condition requiring only
the addition of the sterile liquid carrier, for example, water, for
injections, immediately prior to use. Extemporaneous injection solutions
and suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
The compounds of the present invention may be made into injectable
formulations. The requirements for effective pharmaceutical carriers for
injectable compositions are well known to those of ordinary skill in the
art. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co.,
Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and
ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).
Additionally, the compounds of the present invention may be made into
suppositories by mixing with a variety of bases, such as emulsifying bases
or water-soluble bases. Formulations suitable for vaginal administration
may be presented as pessaries, tampons, creams, gels, pastes, foams, or
spray formulas containing, in addition to the active ingredient, such
carriers as are known in the art to be appropriate.
Methods of Use
In addition, the present invention provides a method of selectively
activating A.sub.3 adenosine receptors in a mammal, which method comprises
acutely or chronically administering to a mammal in need of selective
activation of its A.sub.3 adenosine receptors a therapeutically effective
amount, including a prophylactically effective amount, of a compound which
binds with the A.sub.3 receptor so as to stimulate an A.sub.3
receptor-dependent response.
The method of the present invention has particular usefulness in in vivo
applications. For example, A.sub.3 adenosine receptor agonists can be used
in the treatment of any disease state or condition involving the release
of inositol-1,4,5-triphosphate (IP3), diacylglycerol (DAG), and free
radicals and subsequent arachidonic acid cascades. Thus, high blood
pressure, locomotor hyperactivity, hypertension, acute hypoxia,
depression, and infertility can be treated in accordance with the present
inventive method, wherein one of the above-described compounds is acutely
administered, e.g., within about a few minutes to about an hour of the
onset or realization of symptoms. The method also has utility in the
treatment of chronic disease states and conditions, in particular those
conditions and disease states wherein chronic prophylactic or therapeutic
administration of one of the above-described compounds will prevent the
onset of symptoms or will reduce recovery time. Examples of disease states
and conditions that may be chronically treated in accordance with the
present inventive method include inflammatory disorders, such as vascular
inflammation and arthritis, allergies, asthma, wound healing, stroke,
cardiac failure, acute spinal cord injury, acute head injury or trauma,
seizure, neonatal hypoxia (cerebral palsy; prophylactic treatment involves
chronic exposure through placental circulation), chronic hypoxia due to
arteriovenous malformations and occlusive cerebral artery disease, severe
neurological disorders related to excitotoxicity, Parkinson's disease,
Huntington's chorea, and other diseases of the central nervous system
(CNS), cardiac disease, kidney disease, and contraception.
These compounds can be significant cerebral protectants. As such, the above
compounds can be used to treat and/or protect against a variety of
disorders, including, for example, seizures, transient ischemic shock,
strokes, focal ischemia originating from thrombus or cerebral hemorrhage,
global ischemia originating from cardiac arrest, trauma, neonatal palsy,
hypovolemic shock, and hyperglycemia and associated neuropathies.
The above method is applicable, for example, where a mammal has or is at
risk of having a condition, disorder, or disease state associated with the
cellular release of inositol-1,4,5-triphosphate or diacylglycerol. The
method is also applicable when said mammal has or is at risk for
hyperactivity and said compound in binding to said A.sub.3 adenosine
receptors functions as a locomotor depressant.
The present inventive method is also applicable when said mammal has or is
at risk for hypertension and said compound in binding to said A.sub.3
adenosine receptors functions as a hypotensive agent. The method is also
applicable when said mammal has or is at risk for anxiety and said
compound in binding to said A.sub.3 adenosine receptors functions as an
anxiolytic agent. The method is furthermore applicable when said mammal
has or is at risk for cerebral ischemia and said compound in binding to
said A.sub.3 adenosine receptors functions as a cerebroprotectant. The
method is also applicable when said mammal has or is at risk for seizures
and said compound in binding to said A.sub.3 adenosine receptors functions
as an antiseizure agent.
The present inventive method can be administered chronically as well as
acutely.
The present inventive method includes the administration to an animal, such
as a mammal, particularly a human, in need of the desired A.sub.3
receptor-dependent response of an effective amount, e.g., a
therapeutically effective amount, of one or more of the aforementioned
present inventive compounds or pharmaceutically acceptable salts or
derivatives thereof, alone or in combination with one or more other
pharmaceutically active compounds.
Some of the compounds of the present invention can be utilized as
functionalized congeners for coupling to other molecules, such as amines
and peptides. The use of such congeners provide for increased potency,
prolonged duration of action, specificity of action, and prodrugs. Water
solubility is also enhanced, which allows for reduction, if not complete
elimination, of undesirable binding to plasma proteins and partition into
lipids. Accordingly, improved pharmacokinetics can be realized.
One skilled in the art will appreciate that suitable methods of
administering a compound of the present invention to an animal are
available, and, although more than one route can be used to administer a
particular compound, a particular route can provide a more immediate and
more effective reaction than another route. Accordingly, the
above-described methods are merely exemplary and are in no way limiting.
The dose administered to an animal, particularly a human, in the context of
the present invention should be sufficient to effect a prophylactic or
other therapeutic response in the animal over a reasonable time frame. One
skilled in the art will recognize that dosage will depend upon a variety
of factors including the strength of the particular compound employed, the
age, species, condition, and body weight of the animal, as well as the
severity/stage of the disease or condition. The size of the dose will also
be determined by the route, timing and frequency of administration as well
as the existence, nature, and extent of any adverse side-effects that
might accompany the administration of a particular compound and the
desired physiological effect. It will be appreciated by one of skill in
the art that various conditions or disease states, in particular chronic
conditions or disease states, may require prolonged treatment involving
multiple administrations.
Suitable doses and dosage regimens can be determined by conventional
range-finding techniques known to those of ordinary skill in the art.
Generally, treatment is initiated with smaller dosages, which are less
than the optimum dose of the compound. Thereafter, the dosage is increased
by small increments until the optimum effect under the circumstances is
reached. For convenience, the total daily dosage may be divided and
administered in portions during the day if desired. In proper doses and
with suitable administration of certain compounds, the present invention
provides for a wide range of selective A.sub.3 receptor-dependent
responses. Exemplary dosages range from about 0.01 to about 100 mg/kg body
weight of the animal being treated/day. Preferred dosages range from about
0.1 to about 10 mg/kg body weight/day.
Abbreviations Used in this Application
›.sup.125 I!AB-MECA, N.sup.6
-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide;
CGS 21680,
2-›4-›(2-carboxyethyl)phenyl!ethyl-amino!-5'-N-ethylcarboxamidoadenosine;
CHO, Chinese hamster ovary;
DAST, diethylaminosulfur trifluoride;
DMAP, 4-dimethylaminopyridine;
DMF, N,N-dimethylformamide;
DMSO, dimethylsulfoxide;
EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine;
HMDS, 1,1,1,3,3,3-hexamethyldisilazane;
IB-MECA, N.sup.6 -(3-iodobenzyl)adenosine-5'-N-methyluronamide;
NECA, 5'-N-ethylcarboxamidoadenosine;
PIA, R--N.sup.6 -phenylisopropyladenosine;
THF, tetrahydrofuran; and
TMS-OTf, trimethylsilyl trifluoromethylsulfonate.
EXAMPLES
The following examples further illustrate the present invention and, of
course, should not be construed as in any way limiting its scope. In the
examples, unless otherwise noted, compounds were characterized and
resonances assigned by 300 MHz proton nuclear magnetic resonance mass
spectroscopy using a Varian GEMINI-300 FT-NMR spectrometer. Also, unless
noted otherwise, chemical shifts are expressed as ppm downfield from
tetramethylsilane. Synthetic intermediates were characterized by chemical
ionization mass spectrometry (NH.sub.3) and adenosine derivatives by fast
atom bombardment mass spectrometry (positive ions in a noba or m-bullet
matrix) on a JEOL SX102 mass spectrometer. In the EI mode accurate mass
was determined using a VG7070F mass spectrometer. All adenosine
derivatives were judged to be homogeneous using thin layer chromatography
(silica, 0.25 mm, glass-backed, Alltech Assoc., Deerfield, Ill.;
analytical TLC plates and silica gel (230-400 mesh), VRW, Bridgeport,
N.J.) following final purification. If a mixed solvent was used as the
eluent in chromatographic separation or purification of a compound, the
volume ratio of the solvents is set forth in the illustrative examples
along with the the solvents used. Tables 1 and 2 list the melting point,
elemental analysis, and other data for selected inventive compounds.
Example 1
Preparation of N.sup.6 -(3-Iodobenzyl)-9-Methyladenine (3)
A mixture of 6-chloropurine (1, 100 mg, 0.65 mmol), 3-iodobenzylamine
hydrochloride (192 mg, 0.71 mmol), and triethylamine (0.27 mL, 1.94 mmol)
in absolute ethanol (2 mL) was heated for 24 h at 80.degree. C. After
cooling, the resulting solid was filtered under suction, washed with ethyl
acetate, and dried to give compound 2 (191.3 mg, 84.0%). .sup.1 H NMR
(DMSO-d.sub.6) .delta. 4.67 (br s, 2 H, CH.sub.2), 7.11 (pseudo t, J=7.6
and 7.5 Hz, 1 H, H-16), 7.37 (d, J=7.9 Hz, 1 H, H-17), 7.58 (d, J=7.6 Hz,
1 H, H-15), 7.73 (s, 1 H, H-13), 8.12 and 8.17 (each: s, 1 H, H-8 and
H-2), 8.25 (br s, 1 H, exchangeable with D.sub.2 O, NH), 12.95 (br s, 1 H,
exchangeable with D.sub.2 O, N.sub.9 H).
To a solution of compound 2 (100 mg, 0.28 mmol) in dry DMF (4 mL) were
added potassium carbonate (78.7 mg, 0.57 mmol) and methyl iodide (0.365
mL, 5.7 mmol). The reaction mixture was stirred for 2 h and 20 min at room
temperature. The resulting solid was removed by filtration under suction
and the residue in solution was purified by preparative TLC
(chloroform-methanol, 10:1) to give compound 3 ›R.sub.f =0.51
(chloroform-methanol, 10:1) 25 mg, 24.0%!. .sup.1 H NMR (DMSO-d.sub.6)
.delta. 3.73 (s, 3 H, CH.sub.3), 4.67 (br s, 2 H, CH.sub.2), 7.10 (pseudo
t, J=7.9 and 7.6 Hz, 1 H, H-16), 7.36 (d, J=7.5 Hz, 1 H, H-17), 7.58 (d,
J=7.7 Hz, 1 H, H-15), 7.71 (s, 1 H, H-13), 8.12 and 8.21 (each: s, 1 H,
H-8 and H-2), 8.29 (br s, 1 H, exchangeable with D.sub.2 O, NH).
Example 2
Preparation of 2-Chloro-N.sup.6 -(3-Iodobenzyl)-9-Methyladenine (6)
A solution of 2,6-dichloropurine (4, 2 g, 10.6 mmol), 3-iodobenzylamine
hydrochloride (3.14 g, 11.6 mmol), and triethylamine (4.42 mL, 31.7 mmol)
in ethanol (20 mL) was stirred for 5 days at room temperature. The
resulting solid was filtered, washed with small amount of ethanol, and
dried to give compound 5 (2.32 g, 57.0%). 1H NMR (DMSO-d6) .delta. 4.59
(br d, J=3.5 Hz, 2 H, CH2), 7.13 (pseudo t, J=8.2 and 7.5 Hz, 1 H, H-16),
7.36 (d, J=7.5 Hz, 1 H, H-17), 7.61 (d, J=7.5 Hz, 1 H, H-15), 7.74 (s, 1
H, H-13), 8.14 (s, 1 H, H-8), 8.75 (br s, 1 H, exchangeable with D2O, NH),
13.14 (br s, 1 H, exchangeable with D2O, N.sub.9 H). MS (CI NH.sub.3) m/z
386 (M.sup.+ +1).
A mixture of compound 5 (356 mg, 0.92 mmol), methyl iodide (2.08 mL, 32.4
mmol), and potassium carbonate (256 mg, 1.85 mmol) in DMF (12 mL) was
stirred for 1 h and 40 min at room temperature. After filtration of the
solid, the filtrate was mixed with water (100 mL) and chloroform (30 mL)
and the organic solvent was evaporated. During evaporation of the organic
solvent, a slightly yellow solid formed, which was collected by suction
filtration and dried to yield compound 6 (303 mg, 82.0%). .sup.1 H NMR
(DMSO-d.sub.6) .delta. 3.70 (s, 3 H, CH3), 4.60 (br d, J=5.3 Hz, 2 H,
CH.sub.2), 7.13 (t, J=7.6 Hz, 1 H, H-16), 7.36 (d, J=7.7 Hz, 1 H, H-17),
7.60 (d, J=7.7 Hz, 1 H, H-15), 7.73 (s, 1 H, H-13), 8.14 (s, 1 H, H-8),
8.80 (br s, 1 H, exchangeable with D.sub.2 O, NH).
Example 3
Preparation of 2 -Amino-N.sup.6 -(3-Iodobenzyl)-9 -Methyladenine (9)
A mixture of 6-chloroguanine (7, 100 mg, 0.59 mmol), 3-iodobenzylamine
hydrochloride (175 mg, 0.65 mmol), and triethylamine (0.25 mL, 1.79 mmol)
in ethanol (2 mL) was heated for 94 h at 80.degree. C. The solution was
cooled and diluted with water. The colorless solid that formed was
filtered by suction and dried to give compound 8 (161 mg, 75.0%). .sup.1 H
NMR (DMSO-d.sub.6) .delta. 4.62 (br s, 2 H, CH.sub.2), 5.70 (br s, 2 H,
exchangeable with D.sub.2 O, NH.sub.2), 7.11 (pseudo t, J=7.9 and 7.7 Hz,
1 H, H-16), 7.37 (d, J=7.6 Hz, 1 H, H-17), 7.57 (d, J=7.9 Hz, 1 H, H-15),
7.71 (s, 1 H, H-13), 7.66 (s, 1 H, H-8), 12.09 (br s, 1 H, exchangeable
with D.sub.2 O, NH).
A mixture of compound 8 (100 mg, 0.27 mmol), methyl iodide (0.35 mL, 5.46
mmol), and potassium iodide (75 mg, 0.54 mmol) in dry DMF (4 mL) was
stirred for 1 h and 6 min at room temperature. The solid formed was
removed by suction filtration and the residue in solution was purified by
preparative TLC (chloroform-methanol, 10:1) to give compound 9
(chloroform-methanol, 10:1 R.sub.f =0.46) (3 mg, 2.9%). .sup.1 H NMR
(DMSO-d.sub.6) .delta. 3.54 (s, 3 H, CH.sub.3), 4.61 (br s, 2 H,
CH.sub.2), 5.86 (br s, 2 H, exchangeable with D.sub.2 O, NH.sub.2), 7.10
(t, J=7.7 and 7.6 Hz, 1 H, H-16), 7.27 (d, J=7.3 Hz, 1 H, H-17), 7.36 (d,
J=7.5 Hz, 1 H, H-15), 7.55 and 7.58 (each: s, 1 H, H-13 and H-8), 7.75 (br
s, 1 H, exchangeable with D.sub.2 O, NH).
Example 4
Preparation of 2-Hydrazido-N.sup.6 -(3-Iodobenzyl)-9-Methyladenine (10)
A solution of compound 6 (25 mg, 0.06 mmol) in hydrazine hydrate (1 mL) was
heated for 17 h at 82.degree. C. Water (3 mL) was added, and the colorless
solid formed was filtered by suction and dried to yield compound 10 (19.9
mg, 80.6%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 3.59 (s, 3 H, 9-CH.sub.3),
4.08 (br s, 2 H, exchangeable with D.sub.2 O, NH.sub.2), 4.61 (br s, J=5.3
Hz, 2 H, CH.sub.2), 7.10 (t, J=7.6 Hz, 1 H, H-16), 7.35 (s, 1 H,
exchangeable with D.sub.2 O, NH), 7.39 (d, J=7.6 Hz, 1 H, H-17), 7.57 (d,
J=7.6 Hz, 1 H, H-15), 7.73 (s, 2 H, H-13 and H-8), 7.92 (br s, 1 H,
exchangeable with D.sub.2 O, NH).
Example 5
Preparation of N.sup.6 -(3-Iodobenzyl)-2-Methylamino-9-Methyladenine (11)
A mixture of compound 6 (25 mg, 0.06 mmol) and 2M methylamine in THF (1 mL)
and 40% methylamine in water (1 mL) was stirred for 14 h at 85.degree. C.
After removal of volatiles in vacuo, the residue was triturated with
methanol-water, and the solid that formed was collected by suction
filtration, washed with water (10 mL), and dried to give compound 11 (22
mg, 89.0%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 2.76 (d, J=4.6 Hz, 3 H,
NHCH.sub.3), 3.55 (s, 3 H, 9-CH.sub.3), 4.59 (br s, 2 H, CH.sub.2), 6.28
(br d, J=4.3 Hz, 1 H, exchangeable with D.sub.2 O, NHCH.sub.3), 7.10
(pseudo t, J=7.9 and 7.6 Hz, 1 H, H-16), 7.38 (d, J=7.6 Hz, 1 H, H-17),
7.57 (d, J=7.6 Hz, 1 H, H-15), 7.67 (s, 1 H, H-13), 7.35 (s, 1 H, H-8),
7.83 (br s, 1 H, exchangeable with D.sub.2 O, NH).
Example 6
Preparation of 2-Dimethylamino-N.sup.6 -(3-Iodobenzyl)-9-Methyladenine (12)
A mixture of compound 6 (40 mg, 0.1 mmol), glycine methyl ester (310 mg,
2.47 mmol), and triethylamine (0.7 mL, 5.0 mmol) in DMF (2 mL) was heated
for 22 h at room temperature. After cooling, the mixture was evaporated to
dryness and purified on a silica gel column (chloroform-methanol, 20:1) to
give compound 12 (25 mg, 53.5%) as a colorless solid. .sup.1 H NMR
(DMSO-d.sub.6) .delta. 3.06 (s, 6 H, N(CH.sub.3).sub.2), 3.58 (s, 3 H,
9-CH.sub.3), 4.55 (br s, 2 H, CH2), 7.10 (pseudo t, J=8.0 and 7.6 Hz, 1 H,
H-16), 7.38 (d, J=7.7 Hz, 1 H, H-17), 7.56 (d, J=8.0 Hz, 1 H, H-15), 7.70
(s, 1 H, H-13), 7.77 (s, 1 H, H-8), 7.92 (br s, 1 H, exchangeable with
D.sub.2 O, NH).
Example 7
Preparation of N.sup.6 -(3 -Iodobenzyl)-9-Methyl-2-Propylaminoadenine (13)
A mixture of compound 6 (22.5 mg, 0.056 mmol) and n-propylamine (2 mL) was
stirred at 85.degree. C. for 36 h. After evaporation of volatiles, the
residue was purified by preparative TLC (chloroform-methanol, 20:1) to
give compound 13 (17.3 mg, 72.8%) as a slightly yellow solid. .sup.1 H NMR
(DMSO-d.sub.6) .delta. 0.85 (pseudo t, J=7.5 and 7.3 Hz, 3 H, CH.sub.3),
1.47 (sextet, J=7.2 Hz, 2 H, CH.sub.2), 3.30 (m, 2 H, CH.sub.2), 3.54 (s,
3 H, 9-CH.sub.3), 4.58 (br s, 2 H, CH.sub.2), 6.33 (br s, 1 H,
exchangeable with D.sub.2 O, NH), 7.10 (pseudo t, J=8.0 and 7.7 Hz, 1 H,
H-16), 7.36 (d, J=7.7 Hz, 1 H, H-17), 7.57 (d, J=8.2 Hz, 1 H, H-15), 7.66
(s, 1 H, H-13), 7.72 (s, 1 H, H-8), 7.80 (br s, 1 H, exchangeable with
D.sub.2 O, NH).
Example 8
Preparation of 2-Hexylamino-N.sup.6 -(3-Iodobenzyl)-9-Methyladenine (14)
A mixture of compound 6 (23.5 mg, 0.059 mmol) and n-hexylamine (1 mL) was
heated for 4.5 days at 80.degree. C. After evaporation of volatiles, the
residue was purified by preparative TLC (chloroform-methanol, 20:1) to
give compound 14 (23.5 mg, 86.0%). .sup.1 H NMR (DMSO-d.sub.6) .delta.
0.84 (m, 3 H, CH.sub.3), 1.25 (m, 6 H, CH.sub.2), 1.45 (m, 2 H, CH.sub.2),
3.17 (m, 2 H, CH.sub.2), 3.54 (s, 3 H, 9-CH.sub.3), 4.58 (br s, 2 H,
CH.sub.2), 6.32 (br s, 1 H, exchangeable with D.sub.2 O, NH), 7.09 (pseudo
t, J=7.8 and 7.6 Hz, 1 H, H-16), 7.35 (d, J=7.8 Hz, 1 H, H-17), 7.57 (d,
J=7.7 Hz, 1 H, H-15), 7.66 (s, 1 H, H-13), 7.71 (s, 1 H, H-8), 7.82 (br s,
1 H, exchangeable with D.sub.2 O, NH).
Example 9
Preparation of N.sup.6 -(3-Iodobenzyl)-2-Methoxy-9-Methyladenine (15)
A mixture of compound 6 (21 mg, 0.052 mmol) and sodium methoxide in
methanol (1.5 mg of Na) was heated for 14 h at 85.degree. C. After
evaporation of the solvent, the residue was triturated with methanol-water
to give compound 15 (19 mg, 86.0%). .sup.1 H NMR (DMSO-d6) .delta. 3.64
(s, 3 H, 9-CH.sub.3), 3.81 (s, 3 H, OCH3), 4.59 (br s, 2 H, CH.sup.2),
7.11 (t, J=7.6 Hz, 1 H, H-16), 7.37 (d, J=7.6 Hz, 1 H, H-17), 7.59 (d,
J=7.6 Hz, 1 H, H-15), 7.74 (s, 1 H, H-13), 7.92 (s, 1 H, H-8), 8.37 (br s,
1 H, exchangeable with D.sub.2 O, NH).
Example 10
Preparation of N.sup.6 -(3-Iodobenzyl)-9-Methyl-2-Methylthioadenine (16)
A mixture of compound 6 (24.4 mg, 0.061 mmol) and sodium thiomethoxide (8
mg, 0.1 mmol) in DMF (1.5 mL) was heated for 22 h at 110.degree. C. After
cooling, the reaction mixture was concentrated to dryness and the residue
was purified by a silica gel column chromatography (chloroform-methanol,
20:1) to give compound 16 (13 mg, 52.0%) as a colorless solid. .sup.1 H
NMR (DMSO-d.sub.6) .delta. 3.06 (s, 6 H, N(CH.sub.3).sub.2), 3.56 (s, 3 H,
CH.sub.3), 4.58 (br s, 2 H, CH.sub.2), 7.10 (pseudo t, J=7.9 and 7.6 Hz, 1
H, H-16), 7.38 (d, J=7.6 Hz, 1 H, H-17), 7.56 (d, J=7.9 Hz, 1 H, H-15),
7.76 (s, 1 H, H-13), 7.69 (s, 1 H, H-8), 7.90 (br s, 1 H, exchangeable
with D.sub.2 O, NH).
Example 11
Preparation of N.sup.6 -(3-Iodobenzyl)-9-Methyl-2-(4-Pyridylthio)adenine
(17)
A mixture of compound 6 (20.4 mg, 0.051 mmol) and sodium hydrosulfide
hydrate (11 mg, 0.2 mmol) in pyridine (1.5 mL) was heated for 5 days at
100.degree. C. After cooling, the reaction mixture was concentrated to
dryness and the residue was purified on preparative TLC
(chloroform-methanol, 20:1) to give compound 17 (6.5 mg, 27.4%) as a
yellow solid. .sup.1 H NMR (DMSO-d.sub.6) .delta. 3.78 (s, 3 H, CH.sub.3),
4.70 (br s, 2 H, CH.sub.2), 7.13 (pseudo t, J=7.6 and 7.5 Hz, 1 H, H-16),
7.29 (d, J=7.2 Hz, 2 H, pyr), 7.45 (d, J=7.2 Hz, 1 H, H-17), 7.60 (d,
J=8.2 Hz, 1 H, H-15), 7.86 (s, 1 H, H-13), 8.22 (s, 1 H, H-8), 8.73 (d,
J=7.2 Hz, 2 H, pyr), 9.03 (br s, 1 H, exchangeable with D.sub.2 O, NH).
Example 12
Preparation of N.sup.6 -(3-Iodobenzyl)-9-Hydroxyethyladenine (18)
To a solution of compound 2 (20 mg, 0.056 mmol) and iodoethanol (100 mL) in
dry DMF (0.5 mL) was added anhydrous K.sub.2 CO.sub.3 (50 mg). The mixture
was stirred at room temperature for 10 hours and filtered to remove the
inorganic material. The filtrate was evaporated to dryness and the residue
purified by preparative TLC (CH.sub.2 Cl.sub.2 -MeOH, 10:1,R.sub.f =0.42)
to give compound 18 (27 mg 80%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 3.20
(br s, 1 H, OH), 3.75 (t, J=7 Hz, 2 H, CH.sub.2), 4.21 (t, J=7 Hz, 2 H,
CH.sub.2), 4.67 (br s, 2 H, CH.sub.2), 7.10 (pseudo t, J=7.9 and 7.6 Hz),
1 H, H-16), 7.40 (d, J=7.5 Hz, 1 H, H-17), 7.57 (d, J=7.7 Hz, 1 H, H-15),
7.71 (s, 1 H, H-13), 8.12 and 8,20 (each: s, 1 H, H-8 and H-2), 8.31 (br
s, 1 H, NH).
Example 13
R--N.sup.6 -(3-Iodobenzyl)-9-(2,3-Dihydroxypropyl)adenine (19)
To a solution of compound 2 (60 mg, 0.267 mmol) and
(R)-(-)-2,2-dimethyl-1,3-dioxolan-4-ylmethyl-p-toluenesulfonate (100 mg,
0.35 mmol) in dry DMF (2 mL) was added anhydrous K.sub.2 CO3 (200 mg). The
reaction mixture was heated at 50.degree. C. for 20 h. After cooling to
room temperature, reaction mixture was filtered and the filtrate was
evaporated to dryness. The resulting residue was dissolved in 1N HCl (10
mL) and heated at 80.degree. C. for 1 h. The reaction mixture was cooled
with ice and neutralized by dropwise addition of concentrated NH.sub.4 OH,
and evaporated to dryness. The resulting residue was purified by
preparative TLC (CH.sub.2 Cl.sub.2 -MeOH, 9:1 R.sub.f =0.35) to give
compound 19 (80 mg, 70%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 3.46 (m, 2
H, CH.sub.2), 3.68 (m, 2 H, CH.sub.2), 4.05 (m, 1 H, CH), 4.67 (br s, 2 H,
CH.sub.2), 4.85 (t, 1 H, OH, D.sub.2 O exchangeable), 5.12 (d, 1H, OH,
D.sub.2 O exchangeable), 7.13 (pseudo t, J=7.9 and 7.6 Hz), 1 H, H-16),
7.36 (d, J=7.5 Hz, 1 H, H-17), 7.52 (d, J=7.7 Hz, 1 H, H-15), 7.64 (s, 1
H, H-13), 8.07 and 8.19 (each: s, 1 H, H-8 and H-2), 8.33 (br s, 1 H, NH).
Example 14
Preparation of S--N.sup.6 -(3-Iodobenzyl)-9-(2,3-Dihydroxypropyl)adenine
(20)
Compound 20 was synthesized from
(S)-(+)-2,2-dimethyl-1,3-dioxolan-4-ylmethyl-p-toluenesulfonate following
the procedure described in Example 13. Yield of the purified product, 20,
was 69%. The .sup.1 H NMR in DMSO-d.sub.6 was similar to compound 19.
Example 15
Preparation of N.sup.6 -(3-Iodobenzyladenin-9-yl)acetic Acid (21)
Compound 21 was prepared by following the procedure described in Example
12, starting with compound 2 (0.056 mmol), iodoacetic acid (100 mg), and
K.sub.2 CO.sub.3 (50 mg) in dry DMF (0.5 mL). At the end of the reaction,
the reaction mixture was neutralized by glacial acetic acid before
evaporation to dryness. Yield of Compound 21 after preparative
purification by TLC (CH.sub.2 Cl.sub.2 -MeOH, 9:1 R.sub.f =0.25) was 31 mg
(85%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 4.55 (s, 2 H, CH.sub.2), 4.78
(s, 2 H, CH.sub.2), 7.16 (pseudo t, J=7.9 and 7.6 Hz), 1 H, H-16), 7.42
(d, J=7.5 Hz, 1 H, H-17), 7.61 (d, J=7.7 Hz, 1 H, H-15), 7.79 (s, 1 H,
H-13), 8.40 and 8.45 (each: s, 1 H, H-8 and H-2), 8.90 (br s, 1 H, NH),
12.90 (br s, 1 H, CO.sub.2 H).
Example 16
Preparation of N.sup.6 -(3-Iodobenzyl)-9-(3-Cyanopropyl)adenine (22)
A mixture of N.sup.6 -(3-iodobenzyl)adenine (2, 50 mg, 140 .mu.mol),
4-bromobutyronitrile (300 mg, 2.0 mmol), and potassium carbonate (150 mg,
1.1 mmol) in DMF (2 mL) was stirred for 12 h at 80.degree. C. Following
the addition of 10 mL of half saturated sodium chloride, an oil separated.
The oil was chromatographed on a preparative silica TLC plate
(chloroform:methanol, 95:5, R.sub.f 0.31) to give compound 22 (40 mg,
66%). MS (EI) m/z 418 (M.sup.+), 350, 291, 232, 187.
Example 17
Preparation of 2-Chloro-9-(b-D-erythrofuranoside)-N.sup.6
-(3-iodobenzyl)adenine (28)
To an ice-cold solution of erythrose-1,2,3-triacetate (26, 0.5 gm, 2.03
mmol) in dry acetonitrile (10 mL) were added Compound 5 (0.8 mg, 2.08
mmol), and SnCl.sub.4 (0.8 mg, 3.07 mmol). After warming the reaction
mixture to room temperature, the reaction mixture was heated at 70.degree.
C. for 20 h. The solvent was removed in vacuo, and the resulting residue
was dissolved in concentrated NH.sub.4 OH. This new mixture was refluxed
for 1 h. After evaporation of the volatiles, the residue was purified by
preparative TLC (CH.sub.2 Cl.sub.2 -MeOH, 9.5:0.5, R.sub.f =0.45) to give
Compound 28 (150 mg, 15%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 3.93 (m, 2
H, CH.sub.2), 4.27 (m, 1 H, H-3'), 4.43 (m, 1 H, H-2'), 4.60 (br s, 2 H,
CH.sub.2), 5.31 (d, J=4.5 Hz, 1 H, OH), 4.50 (d, J=4.5 Hz, OH), 6.13 (d,
J=5.9 Hz, 1 H, H-1'), 7.14 (pseudo t, J=7.9 and 7.6 Hz, 1 H, H-5"), 7.34
(d, J=7.5 Hz, 1 H, H-4" or -6"), 7.60 (d, J=7.8 Hz, 1 H, H-4" or -6"),
7.62 (s, 1 H, H-8), 8.37 (s, 1 H, NH), 8.85 (br s, 1 H, N.sup.6 H).
Example 18
Preparation of 9-(.beta.-D-Erythrofuranoside)-2-Methylamino-N.sup.6
-(3-Iodobenzyl)adenine (29)
A solution of Compound 28 (10 mg, 0.021 mmol) in MeOH (1 mL) and 40%
aqueous methylamine (1 mL) was heated in a sealed vessel at 100.degree. C.
for 5 days. After cooling to room temperature, the volatiles were
evaporated and the residue purified by preparative TLC (CH.sub.2 Cl.sub.2
-MeOH, 9.5:0.5) to give Compound 29 as a white solid (9.6 mg, 98%). .sup.1
H NMR (DMSO-d.sub.6) .delta. 2.80 (s, 3 H, NHMe), 3.86 (m, 2 H, CH.sub.2),
4.40 (m, 1 H, H-2'), 4.60 (s, 2 H, CH.sub.2), 5.29 (d, J=4.5 Hz, 1 H, OH),
4.98 (d, J=4.5 Hz, OH), 6.13 (d, J=5.9 Hz, 1 H, H-1'), 7.14 (pseudo t,
J=7.9 and 7.6 Hz, 1 H, H-5"), 7.34 (d, J=7.5 Hz, 1 H, H-4", or -6"), 7.59
(d, J=7.8 Hz, 1 H, H-4" or -6"), 7.60 (d, J=7.8 Hz, 1 H, H-4", or -6"),
7.59 (s, 1 H, H-8), 8.60 (br s, 1 H, N.sup.6 H).
Example 19
Preparation of
2-Chloro-N-(3-Iodobenzyl)-9-(2-Tetrahydrofuryl)-9H-Purin-6-Amine (30)
A solution of 5 (350 mg, 0.91 mmol), 2,3-dihydrofuran (0.38 g, 5.42 mmol),
and 6 drops of ethanesulfonic acid in 30 mL of dry ethyl acetate was
heated for 20 h at 50.degree. C. After cooling the reaction mixture to
room temperature, the volatiles were removed by rotary evaporation and the
residue was purified by preparative silica gel TLC (CH.sub.2 Cl.sub.2
-MeOH, 10:1). After recrystallization from MeOH, Compound 30 (53 mg, 13%)
was obtained as white solid; mp 180.degree.-182.degree. C. .sup.1 H NMR
(DMSO-d.sub.6) .delta. 2.15 (m, 2 H, H-3'), 2.45 (q, J=7.38 Hz, 2 H,
H-2'), 3.92 (q, J=7.38 Hz, 1 H, H-4'), 4.14 (q, J=7.49 Hz, 1 H, H-4'),
4.60 (d, J=5.65 Hz, 2 H, CH.sub.2 -Ph), 6.21 (t, J=5.1 Hz, 1 H, H-1), 7.14
(pseudo t, J=7.9 and 7.6 Hz, 1 H, H-5"), 7.35 (d, J=7.5 Hz, 1 H, H-4", or
-6"), 7.60 (d, J=7.8 Hz, 1 H, H-4" or -6"), 7.62 (d, J=7.8 Hz, 1 H, H-4",
or -6"), 7.74 (s, 1 H, H-8), 8.87 (br s, 1 H, N.sup.6 H).
Example 20
Preparation of 5-O-Benzoyl-3-Deoxy-1,2-Isopropylidene-A-D-Ribofuranoside
(31)
To an ice-cooled solution of
5-O-benzoyl-1,2-isopropylidene-.alpha.-D-xylofuranoside 37 (5.9 g, 0.02
mol) and carbon disulfide (6.03 mL, 0.1 mol) in anhydrous THF (60 mL) was
added 60% sodium hydride in mineral oil (1.6 g, 0.04 mol) at once under
N.sub.2. The reaction mixture was stirred for 50 min. at 0.degree. C. and
methyl iodide (25.7 mL, 0.4 mol) was added. After stirring for 1 h at
0.degree. C., the reaction mixture was neutralized with glacial acetic
acid until the precipitate completely dissolved, and the solution was
concentrated to dryness in vacuo. The residue was dissolved in ethyl
acetate and filtered through a short silica gel column (hexanes-ethyl
acetate, 10:1) to give the xanthate as a brown thick syrup.
A mixture of the xanthate, tributyltin hydride (7.6 mL, 0.029 mol), and
triethylborane (28.6 mL, 0.029 mol) in benzene was stirred for 4 h at room
temperature. After the reaction mixture was concentrated to dryness, the
residue was purified on a silica gel column (hexanes-ethyl acetate,
100:1-followed by 10:1-followed by 3:1) to give compound 31 (1.67 g, 30%).
.sup.1 H NMR (CDCl.sub.3) .delta. 1.27 (s, 3 H, isopropylidene), 1.47 (s,
3 H, isopropylidene), 1.69 (td, J=13.1 and 4.8 Hz, 1 H, H-3b), 2.12 (dd,
J=13.3 and 4.2 Hz, 1 H, H-3a), 4.29 (dd, J=12.1 and 6.0 Hz, 1 H, H-5b),
4.50 (m, 2 H, H-4 and H-5a), 4.72 (t, J=4.2 Hz, 1 H, H-2), 5.81 (d, J=3.7
Hz, 1 H, H-1), 7.35-8.01 (m, 5 H, Bz).
Example 21
Preparation of 3-Deoxy-1,2-Isopropylidene-D-Ribofuranoside (32)
A mixture of compound 31 (1.67 g, 6 mmol) and methanolic ammonia (50 mL,
saturated at 0.degree. C.) was stirred for 5 days at room temperature.
After the reaction mixture was concentrated to dryness, the residue was
purified on a silica gel column (hexanes-ethyl acetate, 100:1-followed by
1:1) to give compound 32 (0.83 g, 79%) as a colorless solid. .sup.1 H NMR
(CDCl.sub.3) .delta. 1.26 (s, 3 H, isopropylidene), 1.45 (s, 3 H,
isopropylidene), 1.66-1.83 (m, 1 H, H-3b), 1.95 (dd, J=13.4 and 4.6 Hz, 1
H, H-3a), 3.50 (m, 1 H, H-5b), 3.83 (dm, J=12.1 Hz, 1 H, H-5a), 4.28 (dm,
J=10.1 Hz, 1 H, H-4), 4.70 (pseudo t, J=4.2 and 4.1 Hz, 1 H, H-2), 5.76
(d, J=3.6 Hz, 1 H, H-1).
Example 22
Preparation of 3-Deoxy-1,2-Isopropylidene-.alpha.-D-5-Ribofuronic Acid (33)
A mixture of compound 32 (0.503 g, 2.89 mmol), ruthenium oxide (38 mg), and
sodium periodate (2.47 g, 11.6 mmol) in 14 ml of a solvent mixture
containing acetonitrile, chloroform, and water (2:2:3 vol. ratio) was
stirred vigorously for 4 h at room temperature. After the aqueous and
organic layers were separated, the aqueous layer was extracted with
chloroform (3.times.50 mL) and combined organic layer and the chloroform
extracts were combined and washed with brine, dried over anhydrous
MgSO.sub.4, filtered, concentrated to dryness, and dried in vacuo to give
compound 33 (0.537 g) as a solid. .sup.1 H NMR (CDCl.sub.3) .delta. 1.28
(s, 3 H, isopropylidene), 1.46 (s, 3 H, isopropylidene), 1.91 (td, J=12.3
and 4.3 Hz, 1 H, H-3b), 2.48 (dd, J=13.6 and 5.2 Hz, 1 H, H-3a), 4.70 (m,
2 H, H-2 and H-4), 5.89 (d, J=3.3 Hz, 1 H, H-1).
Example 23
Preparation of Methyl 3-Deoxy-1,2-Isopropylidene-.alpha.-D-Ribofuronamide
(35)
A mixture of compound 33 (0.48 g, 2.55 mmol), EDAC (1.226 g, 6.42 mmol),
and DMAP (0.031 g, 0.25 mmol) in anhydrous methanol (10 mL) was stirred
for 24 h at room temperature. After the reaction mixture was concentrated
to dryness, the residue was dissolved in chloroform (30 mL) and water (20
mL). The organic layers and aqueous were separated and the aqueous layer
was extracted with chloroform (3.times.30 mL). The organic layer and the
Chloroform extracts were combined and washed with brine, dried over
anhydrous MgSO.sub.4, filtered, and concentrated to dryness. The residue
was purified on a silica gel column (chloroform-methanol, 20:1) to give
compound 34 (0.217 g, 42%).
A solution of compound 34 (217 mg, 1.07 mmol) and 2M methylamine in THF (5
mL) was heated for 24 h at 55.degree. C. in a sealed tube. After the
reaction mixture was concentrated to dryness, the residue was dried in
vacuo to give compound 35 (216 mg, 99%) as needles. .sup.1 H NMR
(CDCl.sub.3) .delta. 1.27 (s, 3 H, isopropylidene), 1.44 (s, 3 H,
isopropylidene), 1.69-1.78 (m, 1 H, H-3b), 2.53 (dd, J=13.7 and 5.2 Hz, 1
H, H-3a), 2.77 (d, J=4.9 Hz, 3 H, NHCH.sub.3), 4.59 (dd, J=11.1 and 5.2
Hz, 1 H, H-4), 4.69 (t, J=4.0 Hz, 1 H, H-2), 5.81 (d, J=3.5 Hz, 1 H, H-1),
6.42 (br s, 1 H, NH). Anal. calcd. for C.sub.9 H.sub.15 N.sub.1 O.sub.4 :
C, 53.72; H, 7.51; N, 6.96. Found C, 53.97; H, 7.65; N, 6.93.
Example 24
Preparation of Methyl 3-Deoxy-1,2-Diacetyl-.alpha.-D-Ribofuronamide (36)
A mixture of compound 35 (189 mg, 0.94 mmol), conc. sulfuric acid (0.276
mL, 5.18 mmol), and acetic anhydride (0.93 mL, 9.86 mmol) in glacial
acetic acid (4.68 mL) was stirred for 18 h at room temperature. The
mixture was cooled in an ice-bath, and saturated NaHCO.sub.3 solution (10
mL) and methylene chloride (10 mL) were added slowly and stirred for 10
min. The organic and aqueous layers were separated, and the aqueous layer
was extracted with methylene chloride (3.times.30 mL). The organic layer
and the methylene chloride extracts were combined, washed with saturated
NaHCO.sub.3 and brine, dried over anhydrous MgSO.sub.4, filtered,
concentrated to dryness, and dried in vacuo to yield crude compound 36
(184 mg, 80%) as a yellow syrup. .sup.1 H NMR (CDCl.sub.3) .delta. 2.00
and 2.03 (each: s, 3 H, OAc), 2.25-2.35 (m, 1 H, H-3b), 2.40-2.47 (m, 1 H,
H-3a), 2.77 (d, J=5.0 Hz, 3 H, NHCH.sub.3), 4.68 (m, 1 H, H-4), 5.12 (d,
J=4.8 Hz, 1 H, H-2), 6.12 (s, 1 H, H-1), 6.35 (br d, J=4.5 Hz, 1 H, NH).
Anal. Calcd for C.sub.10 H.sub.15 N.sub.1 O.sub.6 : C, 48.98; H, 6.17; N,
5.71. Found C, 58.94; H, 6.06; N, 5.42.
Example 25
Preparation of
9-(2-Acetyl-3-Deoxy-.beta.-D-5-Methyl-Ribofuronamido)-2-Chloro -N.sup.6
-(3-Iodobenzyl)adenine (37)
A mixture of 2-chloro-N.sup.6 -(3-iodobenzyl)adenine 5 (163 mg, 0.42 mmol),
ammonium sulfate (catalytic amount), and HMDS (10 mL) was refluxed for 2 h
under N.sub.2. The reaction mixture was concentrated to dryness in vacuo
with exclusion of moisture. The resulting white solid 36a was dissolved in
dry 1,2-dichloroethane (1 mL), and a solution of compound 36 (75 mg, 0.3
mmol) in dry 1,2-dichloroethane (2 mL) and TMS triflate (0.082 mL, 0.42
mmol) were added. The reaction solution was stirred for 1.5 h at room
temperature and refluxed for 17 h at 90.degree. C. under N.sub.2.
Saturated NaHCO.sub.3 (10 mL) and methylene chloride (10 mL) were added
and stirred for 15 min. The aqueous and organic layers were separated and
the aqueous layer was extracted with methylene chloride (3.times.30 mL).
The organic layer and extracts were washed with brine, dried over
anhydrous MgSO.sub.4, filtered, and concentrated to dryness. The residue
was purified by preparative TLC (chloroform-methanol, 20:1) to give
compound 37 (71 mg, 42%). .sup.1 H NMR (CDCl.sub.3) .delta. 2.06 (s, 3 H,
OAc), 2.50 and 2.75 (each: m, 1 H, H-3'), 2.89 (d, J=4.7 Hz, 3 H,
NHCH.sub.3), 4.70 (m, 3 H, H-4' and CH.sub.2), 5.31 (m, 1 H, H-2'), 5.85
(d, J=3.2 Hz, 1 H, H-1'), 6.31 (br s, 1 H, NH), 7.02 (pseudo t, J=7.8 and
7.6 Hz, 1 H, Bn), 7.29 (d, J=7.6 Hz, 1 H, Bn), 7.58 (d, J=7.8 Hz, 1 H,
Bn), 7.67 and 7.72 (each: s, 1 H, H-8 and Bn), 7.84 (br s, 1 H, NH); UV
(MeOH) l.sub.max 271.5 nm.
Example 26
Preparation of
2-Chloro-9-(3-Deoxy-.beta.-D-5-Methyl-Ribofuronamido)-N.sup.6
-(3-Iodobenzyl)adenine (38)
A mixture of compound 37 (15 mg, 0.027 mmol) and NH.sub.3 /MeOH (1.5 mL)
was stirred for 18 h at room temperature. After the reaction mixture was
concentrated to dryness, the residue was purified a silica gel column
chromatography (chloroform-methanol, 20:1) to give compound 38 (6.22 mg,
43%) as a slightly yellow solid. .sup.1 H NMR (DMSO-d.sub.6) .delta.
2.15-2.23 and 2.26-2.35 (each: m, 1 H, H-3), 2.65 (d, J=4.3 Hz, 3 H,
NHCH.sub.3), 4.55-4.68 (m, 4 H, H-2, H-4, CH.sub.2), 5.83 (d, J=3.9 Hz, 1
H, OH, exchangeable with D.sub.2 O), 5.90 (s, 1 H, H-1), 7.13 (t, J=7.6
Hz, 1 H, Bn), 7.37 (d, J=7.6 Hz, 1 H, Bn), 7.61 (d, J=7.7 Hz, 1 H, Bn),
7.75 (s, 1 H, Bn), 8.14 (br s, 1 H, NHCH.sub.3), 8.59 (s, 1 H, H-8), 8.95
(br t, J=5.7 Hz, 1 H, NHCH.sub.2).
Example 27
Preparation of
2-Chloro-9-(2,3-Dideoxy-.beta.-D-5-Methyl-Ribofuronamido)-N.sup.6
-Benzyladenine (40)
A mixture of compound 38 (58.55 mg, 0.11 mmol), phenoxythiocarbonyl
chloride (0.027 mL, 0.19 mmol), DMAP (35.7 mg, 0.29 mmol) in dry
acetonitrile (1.5 mL) was stirred for 6.5 h at room temperature. After the
reaction mixture was concentrated to dryness, the residue was purified by
preparative TLC (chloroform-methanol, 20:1) to give compound 39 as a
glassy solid.
A mixture of compound 39, 1.0M triethylborane in hexanes (0.28 mL, 0.28
mmol), and tributyltin hydride (0.074 mL, 0.28 mmol) in benzene was
stirred for 2.5 h at room temperature. After the reaction mixture was
concentrated to dryness, the residue was purified by preparative TLC
(chloroform-methanol, 20:1) to yield compound 40 (11 mg, 23%) as a
colorless solid. .sup.1 H NMR (CDCl.sub.3) .delta. 2.24-2.60 (m, 4 H, H-2'
and H-3'), 2.89 (d, J=4.8 Hz, 3 H, NHCH.sub.3), 4.53 (dd, J=8.5 and 4.8
Hz, 1 H, H-4'), 4.76 (br s, 2 H, CH.sub.2), 6.01 (pseudo t, J=6.8 and 5.9
Hz, 1 H, H-1'), 6.24 (br s, 1 H, NH), 7.23-7.31 (m, 4 H, Bn), 7.66 (s, 1
H, H-8), 7.83 (br s, 1 H, NH).
Example 28
Preparation of
2-Chloro-9-(3-Deoxy-2-Methanesulfonyl-.beta.-D-5-Methyl-Ribofuronamido
)-N.sup.6 -(3-Iodobenzyl)adenine (41)
Methanesulfonyl chloride (0.05 mL, 0.65 mmol) was added to a solution of
compound 38 (100 mg, 0.18 mmol) in dry pyridine (2 mL) and methylene
chloride (2 mL) and the reaction mixture was stirred for 1.5 h at room
temperature. After rotary evaporation of the solvents, the residue was
purified on a silica gel column (chloroform-methanol, 20:1) to give
compound 41 (87.5 mg, 78%) as a colorless foam. .sup.1 H NMR (CDCl.sub.3)
.delta. 2.66 (ddd, J=11.1, 7.5, and 3.9 Hz, 1 H, H-3'a), 2.86 (m, 1 H,
H-3'b), 2.89 (d, J=5.0 Hz, 3 H, NHCH.sub.3), 3.03 (s, 3 H, OSO.sub.2
CH.sub.3), 4.72 (m, 3 H, H-4' and CH.sub.2), 5.39 (m, 1 H, H-2'), 6.05 (d,
J=3.1 Hz, 1 H, H-1'), 6.31 (br s, 1 H, NH), 7.05 (pseudo t, J=7.8 and 7.6
Hz, 1 H, Bn), 7.28 (d, J=7.7 Hz, 1 H, Bn), 7.58 (d, J=7.7 Hz, 1 H, Bn),
7.67 and 7.77 (each: s, 1 H, H-8 and Bn), 8.65 (br s, 1 H, NH).
Example 29
Preparation of
2-Chloro-9-(2'-Azido-2',3'-Dideoxy-.beta.-D-5'-Methyl-Arabinofuronamido
)-N.sup.6 (3-Iodobenzyladenine) (42)
A mixture of compound 41 (56.6 mg, 0.12 mmol) and sodium azide (83 mg, 1.26
mmol) in anhydrous DMF (2.5 mL) was heated for 41 h at 100.degree. C.
Diethyl ether (30 mL) and water (25 mL) were added, shaken the organic and
aqueous and layers were separated. The aqueous layer was extracted with
ether (3.times.30 mL) and the organic layer and the ether extracts were
combined, washed with brine (30 mL), dried over anhydrous MgSO.sub.4,
filtered, and concentrated to dryness. The residue obtained was purified
by preparative TLC (chloroform-methanol, 20:1, R.sub.f =0.29) to give
compound 42 (22 mg, 34%) as a colorless solid. .sup.1 H NMR (CDCl.sub.3)
.delta. 2.61-2.87 (m, 2 H, H-3'), 2.89 (d, J=5.0 Hz, 3 H, NHCH.sub.3),
4.37 (dd, J=11.1 and 5.2 Hz, 1 H, H-2'), 4.56 (dd, J=8.5 and 6.1 Hz, 1 H,
H-4'), 4.71 (br s, 2 H, CH.sub.2), 6.18 (br s, 1 H, NH), 6.19 (d, J=4.9
Hz, 1 H, H-1'), 7.05 (t, J=7.7 Hz, 1 H, Bn), 1 H, NH), 7.30 (d, J=6.8 Hz,
1 H, Bn), 7.43 (br s, 1 H, NH), 7.59 (d, 7.6 Hz, 1 H, Bn), 7.68 (s, 1 H,
Bn), 7.81 (s, 1 H, H-8).
Example 30
Preparation of
2-Chloro-9-(2'-Amino-2',3'-dideoxy-.beta.-D-5'-methyl-Arabinofuronamido)N.
sup.6 -(3-Iodobenzyl)adenine (43)
A solution of compound 42 (15 mg, 0.027 mmol) and triphenylphosphine (78
mg, 0.3 mmol) in dry THF (2 mL) was stirred for 3 days at room
temperature. Water (0.5 mL) and methanolic ammonia (5 mL) were added and
the reaction mixture was stirred for 21 h at room temperature. After the
reaction mixture was concentrated to dryness, the residue was purified by
preparative TLC (chloroform-methanol, 10:1) to give compound 43 (6 mg,
43%) as a colorless solid. .sup.1 H NMR (DMSO-d.sub.6) .delta. 2.01 (m, 1
H, H-3'a), 2.45 (m, 1 H, H-3'b), 2.64 (d, J=4.7 Hz, 3 H, NHCH.sub.3), 3.80
(m, 1 H, H-2'), 4.40 (pseudo t, J=8.7 and 7.5 Hz, 1 H, H-4'), 4.63 (br s,
2 H, CH.sub.2), 6.09 (d, J=5.7 Hz, 1 H, H-1'), 7.13 (pseudo t, J=8.2 and
7.7 Hz, 1 H, Bn), 7.37 (d, J=7.5 Hz, 1 H, Bn), 7.61 (d, J=8.0 Hz, 1 H,
Bn), 7.75 (s, 1 H, Bn), 8.11 (br s, 2 H, NH.sub.2, exchangeable with
D.sub.2 O), 8.52 (s, 1 H, H-8), 8.88 (br s, 1 H, NH).
Example 31
Preparation of
2-Chloro-9-(2',3'-Dideoxy-2'-Fluoro-.beta.-D-5'-Methyl-Arabinofuronamido)-
N.sup.6 -(3-Iodobenzyl)adenine (44)
To a solution of 2-Cl-IB-MECA (20 mg, 0.04 mmol) in dry dichloromethane
(0.5 mL) at -78.degree. C. was added 50 mL of DAST. After stirring at
-78.degree. C. for 2 h, the reaction mixture was warmed to room
temperature over a period of 1 h and quenched by adding methanol (0.5 mL)
and solid K.sub.2 CO.sub.3 (2 mg). The solvent was removed by evaporation,
and the residue was purified by preparative TLC (CH.sub.2 Cl.sub.2 -MeOH,
9.5:0.5 R.sub.f =0.3) to give 44, (10 mg, 50%). .sup.1 H NMR
(DMSO-d.sub.6) .delta. 2,75 (m, 2 H, H-3'), 3.31 (s, 3 H, NHMe), 4.65 (br
s, 2 H, CH.sub.2), 4.75 (m, 1 H, H-2'), 5.51 (d, J=3.6 Hz, H-4'), 6.21 (d,
J=4.0 Hz, 1 H, H-1'), 7.13 (pseudo t, J=7.9 and 7.6 Hz, 1 H, H-5"), 7.36
(d, J=7.5 Hz, 1 H, H-4" or -6"), 7.61 (d, J=7.8 Hz, 1 H, H-4" or -6"),
7.60 (s, 1 H, H-8), 8.35 (s, 1 H, NH), 8.90 (br s, 1 H, N.sup.6 H).
Example 32
Preparation of
2-Chloro-9-(3,5-1,1,3,3-Tetraisopropyldisiloxyl-.beta.-D-5-Ribofuranosyl)-
N.sup.6 -(3-Iodobenzyl)adenine (45)
To a solution of 2-chloro-N.sup.6 -(3-iodobenzyl)adenosine 5 (300 mg, 0.58
mmol) in dry pyridine (9 mL) was added
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.41 mL, 1.28 mmol) at room
temperature and the reaction mixture was stirred for 2.5 h at room
temperature. The solvent was removed by evaporation, and, after workup,
the residue was purified on a silica gel column (chloroform-methanol,
100:1) to give compound 45(375 mg, 91%) as a colorless foam. .sup.1 H NMR
(DMSO-d.sub.6) .delta. 0.91-1.18 (m, 28 H, isopropyl), 3.17 and 3.49
(each: s, 1 H), 4.03 (m, 3 H), 4.52 (d, J=5.3 Hz, 1 H), 4.70 (br s, 2 H),
5.01 (m, 1 H), 5.83 (s, 1 H), 6.15 (br s 1 H), 7.01 (t, J=7.6 Hz, 1 H,
Bn), 7.28 (d, J=7.6 Hz, 1 H, Bn), 7.55 (d, J=7.7 Hz, 1 H, Bn), 7.66 (s, 1
H, Bn), 7.78 (s, 1 H, H-8).
Example 33
Preparation of
2-Chloro-9-(2',3'-O-Thiocarbonyl-.beta.-D-5-Methyl-Ribofuronamido
)-N.sup.6 -(3-Iodobenzyl)adenine (46)
To a solution of 2-Cl-IB-MECA (10 mg, 0.02 mmol) in dry DMF (0.5 mL) were
added 1,1-thiocarbonyl diimidazole (30 mg 0.17 mmol) and DMAP (2 mg). The
resulting mixture was stirred overnight at room temperature. After removal
of DMF by high vacuum rotary evaporation, the residue was purified by
preparative TLC (CH.sub.2 Cl.sub.2 -MeOH, 9.5:0.5 R.sub.f =0.6) to give
46, (8.6 mg, 80%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 2.73 (d, J=4.3 Hz,
3 H, NHMe), 4.21 (m, 1 H, H-3'), 4.62 (br s, 2 H, CH.sub.2), 5.09 (s, 1 H,
H-4'), 5.95 (m, 1 H, H-2'), 6.31 (d, J=7.3 Hz, 1 H, H-1'), 7.14 (pseudo t,
J=7.9 and 7.6 Hz, 1 H, H-5"), 7.40 (d, J=7.6 Hz, 1 H, H-4" or -6"), 7.60
(d, J=7.8 Hz, 1 H, H-4" or -6"), 7.76 (s, 1 H, H-2"), 8.27 (br d, J=4.3
Hz, 1 H, exchangeable with D.sub.2 O, NH), 8.49 (s, 1 H, H-8), 9.02 (br t,
J=6.2 and 5.7 Hz, 1 H, exchangeable with D.sub.2 O, N.sup.6 H).
Example 34
Preparation of (1R,4S)-4-(2,6-Dichloro-9H-Purin-9-yl) Cyclopent-2-en-1-ol
(48)
To a solution of 2,6-dichloropurine (2 g, 10.64 mmol) in dry DMSO (25 mL)
was added sodium hydride (60% suspension in mineral oil, 0.42 g, 10.64
mmol). The reaction mixture was stirred at the room temperature for 30
min, followed by the addition of tetrakis (triphenylphosphine)-palladium
(0.5 g, 0.22 mmol), triphenylphosphine (0.25 g, 0.95 mmol) and a solution
of (+)-47.sup.20 (1.66 g, 11.70 mmol) in dry THF (25 mL). This mixture was
stirred at 50.degree. C. for 20 h. The volatiles were removed by rotary
evaporation in vacuo at 50.degree. C. The residue was slurried in CH.sub.2
Cl.sub.2 (50 mL) and filtered to remove insoluble solids. The filtrate
that resulted was washed with brine (2.times.50 mL), dried over anhydrous
MgSO.sub.4 and evaporated to dryness. The residual oil was purified by
flash chromatography on silica gel by eluting first with AcOEt to remove
the non-polar impurities and then with AcOEt-MeOH (9:1). The product
containing fractions were evaporated to dryness to give 48 (3.55 g, 89%)
as colorless foam. .sup.1 H NMR (DMSO-d.sub.6) .delta. 1.62-2.7 (m, 2 H,
H-5'), 3.26 (m, 3 H, OH), 3.65 (m, 1 H, H-1'), 4.58 (m, 3 H, H-2', H-3',
H-4'), 8.34 (m, 1 H, H-8).
Example 35
Preparation of (1S, 2R, 3S, 4R)-4-(6-Amino-2-Chloro-9H-Purin-9-yl)
Cyclopentane-1,2,3-triol (50)
To a solution of 48 (1 g, 3.70 mmol) in THF-H.sub.2 O (10:1, 50 mL) was
added a 60% aqueous solution of N-Methylmorpholine N-oxide (1.2 mL, 1.14
mmol) and then osmium tetroxide (30 mg). The reaction mixture was stirred
at room temperature for 24 h. The solvent was removed by rotary
evaporation and the residue was co-evaporated with EtOH (3.times.50 mL) to
give a gummy material. This residue was dissolved in MeOH presaturated
with anhydrous ammonia (50 mL) and stirred in a sealed tube at room
temperature for 5 days. Volatiles were removed by rotary evaporation and
the residue was subjected to column chromatography on silica gel
(MeOH--CH.sub.2 Cl.sub.2, 9:1) to give 50 (800 mg, 80%) as white solid: mp
180.degree. C. (dec.); .sup.1 H NMR (DMSO-d.sub.6) .delta. 1.77-190 (m, 1
H, H-5'), 2.50 (m, 1 H, H-5') 3.75 (m, 1 H, H-2'), 3.89 (m, 1 H, H-1'),
4.40-4.52 (m, 1 H, H-3'), 4.62-4.81 (m, 1 H, H-4'), 4.87 (d, 1 H, J=3.6
Hz, OH), 5.01 (d, 1 H, J=6.6 Hz, OH), 5.36 (d, 1 H, J=4.8 Hz, OH), 7.22
(s, 2 H, NH.sub.2), 8.11 (s, 1 H, H-8).
Example 36
Preparation of (1S, 2R, 3S,
4R)-4-(6-Amino-2-Phenylethylamino-9H-Purin-9-yl)Cyclopentane-1,2,3-triol
(51)
To a suspension of compound 50 (300 mg, 1.05 mmol) in absolute EtOH (10 mL)
was added phenylethylamine (300 mg, 2.45 mmol). This mixture was heated at
90.degree. C. for 3 days. The solvent was removed by rotary evaporation,
and the residue was subjected to column chromatography on silica gel
(CH.sub.2 Cl.sub.2 -MeOH) to give compound 51 (272 mg, 70%) as a yellow
solid: mp. 200.degree. C. (dec.); .sup.1 H NMR (DMSO-d.sub.6) .delta.
1.77-1.90 (m, 1 H, H-5'), 2.50-2.60 (m, 1 H, H-5'), 2.8 (m, 4 H, 2 x
CH.sub.2), 3.75 (m, 1 H, H-2'), 3.89 (m, 1 H, H-1'), 4.40-4.52 (m, 1 H,
H-3'), 4.62-4.81 (m, 1 H, H-4'), 4.87 (d, 1 H, J=3.6 Hz, OH), 5.01 (d, 1
H, J=6.6 Hz, OH), 5.36 (d, 1 H, J=4.8 Hz, OH), 7.22 (s, 2 H, NH.sub.2),
7.48 (m, 5 H, Ph), 8.48 (s, 1 H, H-8).
Example 37
Preparation of Methyl
2,3-O-Isopropylidene-N,4-Dimethyl-.beta.-D-Ribofuranosiduronamide (53)
A solution of methyl ester 52 (502 mg, 2.04 mmol) (Johnson et al., J. Org.
Chem., 59, 5854-5855 (1994)) in MeOH (30 mL) was saturated with gaseous
methylamine and stirred until TLC (2:1, hexane:EtOAc) showed that the
reaction was complete. The reaction mixture was concentrated, and the
residue chromatographed on silica gel (1.5:1, hexane:EtOAc) to give 502 mg
(100%) of 53 as a clear oil: ›a!.sub.D.sup.23 -45.0.degree. (c 1.05
CH.sub.2 Cl.sub.2); .sup.1 H NMR (300 MHz, CDCl.sub.3) d 6.56 (br s, 1H),
5.10 (d, 1H, J=5.9 Hz), 4.93 (s, 1H), 4.51 (d, 1H, J=5.9 Hz), 3.39 (s,
3H), 2.79 (d, 3H, J=5.0 Hz), 1.47 (s, 3H), 1.44 (s, 3H), 1.30 (s, 3H);
.sup.13 C NMR (75 MHz, CDCl.sub.3) .delta. 174.55, 112.53, 110.25, 89.33,
85.15, 82.35, 55.96, 26.16, 25.98, 24.76, 20.96.
Example 38
Preparation of Methyl
2,3-O-Diacetyl-N,4-Dimethyl-.beta.-D-Ribofuranosiduronamide (54)
To a solution of the acetonide 53 (502 mg, 2.04 mmol) in MeOH (60 mL) was
added concentrated HCl (0.1 mL). The solution was stirred for 18 h and
then concentrated under reduced pressure. The residue was chromatographed
on silica gel (gradient EtOAc-30% MeOH in EtOAc) to give 105 mg (21%) of
starting acetonide and 310 mg of a mixture of stereo isomeric diols as a
mixture. The diols were then acetylated by mixing with Ac.sub.2 O (0.48
mL) and pyridine (0.85 mL) in CH.sub.2 Cl.sub.2 (30 mL) containing a
catalytic amount of DMAP. Toluene (10 mL) was added to the reaction
mixture which was then concentrated to dryness. The residue was
chromatographed on silica gel (1:1, hexane:EtOAc) to give 380 mg (64%) of
54-b (OMe axial) and 40 mg (7%) of 54-a (OMe equatorial) (81% and 8.5%
yields respectively based on the recovered acetonide). 54-b: oil;
›a!.sub.D.sup.23 -27.5.degree. (c 1.2, CH.sub.2 Cl.sub.2); .sup.1 H NMR
(300 MHz, CDCl.sub.3) .delta. 6.77 (br s, 1H), 5.54 (d, 1H, J=4.8 Hz),
5.19 (dd, 1H, J=3.2, 4.8 Hz), 5.01 (d, 1H, J=3.2 Hz), 3.48 (s, 3H), 2.82
(d, 3H, J=5.0 Hz), 2.10 (s, 3H), 2.05 (s, 3H), 1.46 (s, 3H); .sup.13 C NMR
(75 MHz, CDCl.sub.3) .delta. 173.26, 169.19 (2), 106.54, 85.69, 74.71,
73.75, 56.87, 26.11, 20.56 (2), 20.45. Anal. calcd. for C.sub.12 H.sub.19
NO.sub.7 : C, 49.82; H, 6.62. Found: C, 49.75; H, 6.52. 54-a: mp.
112.degree.-113.degree. C. (CH.sub.2 Cl.sub.2 /hexane); .sup.1 H NMR (300
MHz, CDCl.sub.3) .delta. 6.71 (br s, 1H), 5.64 (d, 1H, J=6.3 Hz), 5.12 (d,
1H, J=4.8 Hz), 4.94 (dd, 1H, J=6.3, 4.8 Hz), 3.42 (s, 3H), 2.81 (d, 3H,
J=5.0 Hz), 2.16 (s, 3H), 2.08 (s, 3H), 1.48 (s, 3H); .sup.13 C NMR (75
MHz, CDCl.sub.3) .delta. 172.92, 169.81, 169.58, 101.35, 85.59, 72.08,
70.78, 55.83, 26.08, 21.14, 20.65, 20.45.
Example 39
Preparation of
2,3-O-Diacetyl-1-(6-Chloro-9H-Purin-9-yl)-1-Deoxy-N,4-Dimethyl-.beta.-D-Ri
bofuranosiduronamide (55)
A suspension of 6-chloropurine (590 mg, 3.82 mmol) in HMDS (6 mL) was
heated to 100.degree. C. until dissolution was complete, ca. 1 h. Toluene
(2 mL) was added and the solution was concentrated under an inert
atmosphere. To remove final traces of HMDS, toluene (2.times.4 mL) was
again added, and the solution was concentrated in a similar manner. The
silylated 6-chloropurine was dissolved in dry CH.sub.3 CN (3 mL): 307 mg
(1.05 mmol) of compound 54-b (dried by azeotropic distillation with
toluene under reduced pressure) in dry CH.sub.3 CN (5 mL) and
trimethylsilyl trifluoromethanesulfonate (0.75 mL) were added, and the
reaction mixture was heated to reflux for 12 h. The two initial nucleoside
products detected by TLC gave way to a single thermodynamic product during
this time. The reaction was cooled and quenched by the addition of
saturated aqueous NaHCO.sub.3 (1 mL) and partitioned between CH.sub.2
Cl.sub.2 (40 mL) and H.sub.2 O (10 mL). The aqueous layer was extracted
with CH.sub.2 Cl.sub.2 (2.times.40 mL). The combined organics were dried
(MgSO.sub.4), filtered, and concentrated under reduced pressure. Column
chromatography on silica gel (1:2, CH.sub.2 Cl.sub.2 : EtOAc) gave 270 mg
(63%) of 6-Cl-purine nucleoside 55 as a faint yellow foam:
›a!.sub.D.sup.23 2.48.degree. (c 1.45, CH.sub.2 Cl.sub.2); .sup.1 H NMR
(300 MHz, CDCl.sub.3) d 8.68 (s, 1H), 8.27 (s, 1H), 7.61 (br q, 1H, J=4.9
Hz), 6.15 (d, 1H, J=7.5 Hz), 6.00 (dd, 1H, J=7.5, 5.0 Hz), 5.82 (d, 1H,
J=5.0 Hz), 2.77 (d, 3H, J=4.9 Hz)), 2.13 (s, 3H), 1.88 (s, 3H), 1.49 (s,
3H); .sup.13 C NMR (75 MHz, CDCl.sub.3) .delta. 171.13, 168.78 (2),
151.80, 151.56, 150.96, 144.67, 133.39, 86.80, 86.07, 73.03, 71.15, 25.99,
20.20, 19.96, 19.75.
Example 40
Preparation of
1-(6-Benzylamino-9H-Purin-9-yl)-1-Deoxy-N,4-dimethyl-.beta.-D-Ribofuranosi
duronamide (56)
The diacetyl nucleoside 54 (195 mg, 0.474 mmol) was selectively deacylated
by treatment with a methanolic solution of NH.sub.3 at 0.degree. C. for 10
min. The solution was evaporated to dryness and to the residue was added a
1:1 solution of t-BuOH and benzylamine (4 mL). This solution was heated at
70.degree. C. for 16 h, and concentrated under reduced pressure (0.1 torr,
40.degree. C.). Chromatography of the residue on silica gel (gradient
30:1-10:1, CH.sub.2 Cl.sub.2 :MeOH) gave 159 mg (84%) of N-benzyladenine
nucleoside 56 as a white solid: ›a!.sub.D.sup.23.varies.-25.7 .degree. (c
1.02, CH.sub.3 OH); .sup.1 H NMR (500 MHz, CD.sub.3 OD, prior H-D
exchange) .delta. 8.29 (s, 1H), 8.13 (s, 1H), 7.35 (d, 2H, J=7.0 Hz), 7.28
(t, 2H, J=7.5 Hz), 7.21 (t, 1H, J=7.5 Hz), 5.97 (d, 1H, J=8.5 Hz), 4.83
(dd, 1H, J=8.5, 5.0 Hz), 4.78 (br s, 2H), 4.30 (d, 1H, J=5.0 Hz), 2.82 (s,
3H), 1.50 (s, 3H); .sup.13 C NMR (125 MHz, CD.sub.3 OD) .delta. 174.98,
154.73, 152.48, 148.33, 140.77, 138.78, 128.13, 127.10, 126.81, 120.10,
88.16, 87.74, 73.75, 71.75, 43.62, 24.90, 18.79.
Example 41
Preparation of
(.+-.)-9-›2.alpha.,3.beta.-Dihydroxy-4.beta.-(N-Methylcarbamoyl)Cyclopent-
1.beta.-yl)!-6-Chloropurine (60)
To a solution of compound 57 (1 g, 7.04 mmol, prepared according to a
procedure reported by Cermak and Vince, Tetrahedron Lett., 22, 2331-2332
(1981)) in dry MeOH (20 mL) was bubbled anhydrous methylamine for 10 min.
The resulting solution was heated at 90.degree. C. in a sealed tube for 20
h. After cooling to room temperature, solvent was removed by rotary
evaporation in vacuo, and the residue was used in next step without
further characterization. To this residue was added
5-amino-4,6-dichloropyrimidine (1.00 g, 6.13 mmol), triethylamine (2 mL),
and n-BuOH (20 mL). The resulting mixture was heated at 100.degree. C.,
under N.sub.2 atmosphere, for 24 hours. Volatiles were evaporated in vacuo
and the residue was dissolved in diethoxymethyl acetate (10 mL). This
mixture was heated at 100.degree. C. for 2 h and then evaporated to
dryness. The residue was dissolved in 1N HCl (10 mL) and stirred at the
room temperature for 3 h. The ice-cold reaction mixture was neutralized
with conc. NH.sub.4 OH and evaporated to dryness. The residue was
subjected to column chromatography on a silica gel column (CH.sub.2
Cl.sub.2 -MeOH, 9.5:0.5) to give 60 (1.3 g, 59% yield based upon 58) as
yellow foam. .sup.1 H NMR (DMSO-d.sub.6) .delta. 2.75 (s, 2 H, CH.sub.2),
3.33 (m, 1 H, H-1'), 3.40 (d, J=4.3 Hz, 3 H, Me), 4.22 (m, 1 H, H-3'),
4.33 (s, 1 H, H-4'), 4.75 (dd, J=4.0 Hz, J=4.3 Hz, 1 H, H-2'), 5.45 (d,
J=6.4 Hz, 1 H, OH-2'), 5.60 (d, J=4.1 Hz, 1 H, OH-3'), 5.60 (d, J=7.4 Hz,
1 H, H-1'), 8.21 (s, 1 H, H-8), 8.50 (br s, 2 H, HN.sup.6), 8.60 (br s, 1
H, NH--Me).
Example 42
Preparation of
(.+-.)-9-›2.alpha.,3.beta.-Dihydroxy-4.beta.-(N-Methylcarbamoyl)Cyclopent-
1.beta.-yl)!-N.sup.6 -(3-Iodobenzyl)-adenine (61)
To a solution of compound 60 (100 mg, 0.32 mmol) in absolute EtOH (10 mL)
was added 3-iodobenzylamine hydrochloride (90 mg, 0.34 mmol) and the
resulting mixture was heated at 90.degree. C. for 24 hours, under a
nitrogen atmosphere. The solvent was removed by evaporation in vacuo and
the residue was purified on a silica gel column (CH.sub.2 Cl.sub.2 -MeOH,
10:0.5) to give compound 61 (140 mg, 85%) as colorless foam. .sup.1 H NMR
(DMSO-d.sub.6) .delta. 2.71 (s, 2 H, CH.sub.2), 3.31 (m, 1 H, H-1'), 3.42
(d, J=4.3 Hz, 3 H, Me), 4.32 (m, 1 H, H-3'), 4.35 (s, 1 H, H-4'), 4.70 (s,
2 H, CH.sub.2 -Ph), 4.74 (dd, J=4.0 Hz, J=4.3 Hz, 1 H, H-2'), 5.45 (d,
J=6.4 Hz, 1 H, OH-2'), 5.60 (d, J=4.1 Hz, 1 H, OH-3'), 5.60 (d, J=7.4 Hz,
1 H, H-1'), 7.13 (t, 1 H, J=7.1 Hz), 7.40 (d, J=7.7, 1 H), 7.60 (d, J=7.6
Hz, 1 H), 8.21 (s, 1 H, H-8), 8.50 (br s, 1 HN.sup.6), 8.60 (br s, 1 H,
NH--Me).
Example 43
Preparation of N.sup.6
-›3-(L-Prolylamino)Benzyl!Adenosine-5'-N-Methyluronamide (65)
20 mg (45.51 .mu.mol) of 2',3'-O-isopropylidene-N.sup.6
-(3-aminobenzyl)adenosine-5-N-methyluronamide 62 (Gallo-Rodriguez et al.
J. Med. Chem., 37, 636-646 (1994), N-t-Boc-L-proline (12 mg, 55.76
.mu.mol), N,N-dicyclohexylcarbodiimide (18.77 mg, 90.98 .mu.mol) and
imidazole (6.2 mg, 91.07 .mu.mol) were dissolved in anhydrous DMF. The
solution was stirred at room temperature for 20 hours in a sealed vessel.
The solvent was evaporated in a rotary evaporator and high vacuum. The
residue was dissolved in hydrochloric acid (1M, 0.5 mL), and the resulting
solution was heated to 60.degree. C. for 40 min. After cooling in an ice
bath, sodium bicarbonate solution was added to neutralize, and the solvent
was removed under vacuum. The residue was subjected to preparative silica
gel TLC (MeOH:CH.sub.2 Cl.sub.2 8:2) to give compound 65 as white solid
(14.2 mg, 63% yield overall). .sup.1 H NMR (DMSO-d.sub.6) .delta. 2.32 (m
2H,CH.sub.2), 2.70(d, J=4.6 Hz, 3H, CH.sub.3), 2.73 (m, 2H, CH.sub.2),
3.79 (m, 2H,CH.sub.2 -N), 4.2 (m, 1H, H-3"), 4.30 (s, 1H, H-4'), 4.60 (m,
2H, H-2'), 4.67 (br s, 2H, N.sup.6 --CH.sub.2 Ph), 5.54 (d, J=6.4 Hz, 1H,
OH-2'), 5.70 (d, J=4.1 Hz, 1H, OH-3'), 5.97 (d, J=7.6 Hz, 1H, H-1'), 7.2
(t, J=7.7, 1H), 7.40 (d, J=7.7 Hz, 1H), 7.60(d, J=7.8, 1H), 7.71 (br s,
1H, NH--CH.sub.2), 7.72 (s, 1H), 8.28 (2 1H, H-2), 8.50 (s, 2H, H-8), 8.56
(br s, 1H, N.sup.6 H--CH.sub.2 Ph), 8.80 (br s, 1H, NHMe) (8.90) (br s,
1H, NH--Ph), High resolution MS (m/z) measured in FAB+ mode: Calcd for
C.sub.23 H.sub.28 N.sub.8 O.sub.5 496.5302, found 496.5306.
Example 44
Preparation of N.sup.6
-›3(.beta.-Alanylamino)Benzyl!Adenosine-5'-Methyluronamide (66)
Compound 62 (40 mg, 91.02 .mu.mol), N.sup.6 -t-Boc-.beta.-alanine (24 mg,
127.42 .mu.mol), and EDAC (30 mg, 156.49 .mu.mol) were dissolved in
anhydrous DMF. The solution was stirred at room temperature for 24 hours
under nitrogen. The solvent was removed under vacuum, and the residue was
dissolved in hydrochloric acid (1N, 1 mL). The resulting mixture was
heated to 60.degree. C. for 40 min. After cooling in an ice bath, conc.
NH.sub.4 OH was added to neutralize. The reaction mixture was loaded on a
small Dowex 50X2-200 (H+) resin column. The column was eluted with water
until eluents were neutral to pH paper. Finally the column was eluted with
1N NH.sub.4 OH, and the product-containing fractions were lyophilized to
give compound 66 as a yellow solid (29.2 mg, 68% overall yield). .sup.1 H
NMR (DMSO-d.sub.6) .delta. 2.63 (m, 2H, CH.sub.2), 2.70 (d, J=4.3 Hz, 3H,
CH.sub.3), 2.75 (m, 2H, CH.sub.2), 4.14 (m, 1H, H-3'), 4.32 (s, 1H, H-4'),
4.59 (dd, J=4.6 Hz, J=7.5 Hz, 1H, H-2'), 4.71 (br s, 2H, N.sup.6
--CH.sub.2 Ph), 5.55 (d, J=6.4 Hz, 1H, OH-2'), 5.68 (d, J=4.1 Hz, 1 H,
OH-3'), 5.95 (d, J=7.4 Hz, 1H, H-1'), 7.10 (t, J=7.7 Hz, 1H), 7.35 (d,
J=7.7 Hz, 1H), 7.58 (d, J=7.8, 1 H), 7.72 (s, 1H), 8.30 (s, 1H, H-1), 8.44
(s, 1H, H-8), 8.56 (br s, 1H, N.sup.6 H--CH.sub.2 Ph), 8.80-89 (m, 2H,
NH--Me & NH--Ph). High resolution MS (m/z) measured in FAB+ mode: Calcd
for C.sub.21 H.sub.26 N.sub.8 O.sub.5 470.4919, found 470.4921.
Example 45
Preparation of N.sup.6
-›3-(N-6-Boc-.beta.-Alanylamino)Benzyl!Adenosine-5'-Methyluronamide (67)
A solution of compound 66 (10 mg, 21.25 .mu.mol), di-tert-butyldicarbonate
(5.5 mg, 25.2 .mu.mol) and triethylamine (20 .mu.L) in anhydrous DMF (0.5
mL) was stirred at room temperature under nitrogen. The solvent was
removed in vacuo, and the residue was purified by preparative silica gel
TLC (CH.sub.2 Cl.sub.2: MeOH 9:1) to give compound 67 as a white solid
(9.7 mg, 80% yield overall). .sup.1 H NMR (DMSO-d.sub.6) .delta. 1.38 (s,
9H, CH.sub.3), 2.71 (m, 2H, CH.sub.2), 3.26 (m, 2H, CH.sub.2), 3.31 (d,
J=4.3 Hz, 3H, CH.sub.3), 4.12 (m, 1H, H-3'), 4.33 (s, 1H, H-4'), 4.60 (dd,
J=4.6 Hz, J=7.5 Hz, 1H, H-2'), 4.70 (br. s, 2H, N.sup.6 --CH.sub.2 PH),
5.53 (d, J=6.4 Hz, 1H, OH-2'), 5.71 (d, J=4.1 Hz, 1 H, OH-3.sup.1), 5.60
(d, J=7.4 Hz, 1H, H-1), 7.13 (t, J=7.7 Hz, 1H), 7.40 (d, J=7.7 Hz, 1H),
7.60 (d, J=7.8, 1H), 7.71 (s, 1H), 8.28 (s, 1H, H-1), 8.42 (s, 1H, H-8),
8.56 (br s, 1H, N.sup.6 H--CH.sub.2 Ph), 8.55 (br s, 1H, NH--Me), 8.90 (br
s, 1H, NH--Ph), 9.80 (br s, 1 H, CH.sub.2 NN--CO). High resolution MS
(m/z) measured in FAB+ mode: Calcd for C.sub.26 H.sub.34 N.sub.8 O.sub.7
570.6103, found 570.6106.
Example 46
Preparation of
6-O-Phenylhydroxylamino)Purine-9-.beta.-Ribofuranoside-5'-N-Methyluronamid
e (71)
Compound 61 (30 mg, 85.04 .mu.mol) and Dowex 30X2-200 (H+ resin, 2 mL, dry
volume in water (3 mL)) were heated at 80.degree. C. for one hour. The
reaction mixture was made slightly basic by adding concentrated NH.sub.4
OH and filtered, and the filtrates were evaporated to dryness. The residue
was co-evaporated few times with absolute ethanol. Thereafter
O-phenylhydroxylamine hydrochloride (18 mg, 124.2 mol) and triethylamine
(23.7 L, 0.70 mmol) were added, and the resulting mixture was heated at
65.degree. C. for 24 hours. The solvent was removed under a stream of
nitrogen, and the residue was purified by TLC (CH.sub.2 CL.sub.2 :MeOH,
8:2) to obtain 17 mg (52%) of the title compound. .sup.1 H NMR (DMSO
-d.sub.6) .delta. 3.26 (d, J=4.3 Hz, 3H, CH.sub.3), 4.22 (m, 1H,
H-3.sup.1), 4.31 (s, 1H, H.sup.1), 4.62 (dd, J=4.6 Hz, J=7.5 Hz, 1H,
H-2.sup.1), 5.53 (d, J=6.4 Hz, 1H, OH-2.sup.1), 5.70 (d, J=4.1 Hz, 1H,
OH-3.sup.1), 5.70 (d, J=7.4 Hz, 1H, H-1.sup.1), 6.90 (m, 3H, Ph), 7.30 (m,
2H, Ph), 8.30 (s, 1H, H-1), 8.41 (s, 1H, H-8), 8.56 (br s, 1H, NH--O),
8.55 (br s, 1H, NH--Me). High resolution Ms (m/z) measured in FAB+ mode:
Calcd for C.sub.17 H.sub.18 N.sub.6 O.sub.5 386.3702, found 386.3705.
Example 47
Preparation of
6-(N'-phenylhydrazinyl)Purine-9-.beta.-Ribofuranoside-5'-N-Methyluronamide
(70)
Compound 61 (30 mg, 85.04 umol), phenylhydrazine (10 mg, 92.5 umol), and
triethylamine (23.7 .mu.L, 0.70 mmol) were dissolved in absolute ethanol
(1 mL). The solution was stirred at 70.degree. C. for 16 hours under
nitrogen. The solvent was evaporated under a stream of nitrogen, and
hydrochloric acid (1N, 1 mL) was added, and the resulting solution was
heated to 60.degree. C. for 40 min. After cooling in an ice bath, sodium
bicarbonate solution was added to neutralize. Volatiles were removed under
vacuum, and the residue was purified by preparative silica gel thin layer
chromatography (CH.sub.2 Cl.sub.2 :MeOH 8:2) to obtain 18 mg (55%) of
compound 70. .sup.1 H NMR (DMSO-d.sub.6) 3.29 (d, J=4.3 Hz, 3H, CH.sub.3),
4.20 (m, 1H, H-3'), 4.35 (s, 1H, H-4'), 4.60 (dd, J=4.6 Hz, J=7.5 Hz, 1H,
H-2'), 5.50 (d, J=6.4 Hz, 1H, OH-2'), 5.68 (d, J=4.1 Hz, 1 H, OH-3'), 5.66
(d, J=7.4 Hz, 1H, H-1'), 6.93 (m, 3H, Ph), 7.31 (m, 2H, Ph), 8.29 (s, 1H,
H-1), 8.43 (s, 1H, H-8), 8.56 (br s, 2H, NH--NH), 8.55 (br s, 1H, NH--Me).
High resolution MS (m/z) measured in FAB+ mode: Calcd for C.sub.17
H.sub.19 N.sub.7 O.sub.4 385.3855, found 385.38550.
Example 48
Preparation of N.sup.6 -(Benzodioxanemethyl)Adenosine Hemihydrate (72)
6-Chloropurine riboside (200 mg, 0.70 mmol) was refluxed in 10 mL ethanol
with 162 mg (0.77 mmol) racemic benzodioxane-2-methylamine and 2 g (2.1
mmol) triethylamine solution (5.3 g triethylamine in 50 g ethanol) for 18
h. After 48 h at -20.degree. C., a white crystalline product was formed
that was collected and dried. Further workup afforded a second crop of the
product, compound 72. Total yield 180 mg (62%). M.p.
176.degree.-178.degree. C. MS (CI), MH.sup.+ =416.
Example 49
Preparation of
1-(6-Furfurylamino-9H-Purin-9-yl)-1-Deoxy-N-Methyl-.beta.-D-Ribofuranosidu
ronamide (74)
To a solution of
2',3'-isopropylidene-1-(6-chloro-9H-purin-9-yl)-1-deoxy-N-methyl-.beta.-D-
ribofuranosiduronamide (Gallo-Rodriguez et al.) (50 mg, 0.16 mmol) in
absolute EtOH (5 mL) was added furfurylamine (20 mg, 0.21 mmol). This
mixture was heated at 90.degree. C. for 20 h. After cooling to room
temperature, solvent was removed by rotary evaporation, and the residue
was dissolved in 0.5N HCl and heated at 60.degree. C. for 1 h. After
cooling to 0.degree. C. in an ice-bath, the reaction mixture was
neutralized with concentrated NH.sub.4 OH and evaporated to dryness. The
residue was purified by preparative thin-layer chromatography on silica
gel to give compound 74 (21 mg, 40%) as a white solid: .sup.1 H NMR
(DMSO-d.sub.6) .delta. 3.32 (d, J=4.3 Hz, 3 H, Me), 4.12 (m, 1 H, H-3'),
4.33 (s, 1 H, H-4'), 4.60 (dd, J=4.6 Hz, J=4.3 Hz, 1 H, H-2'), 4.70 (br s,
2 H, N.sup.6 --CH.sub.2 --), 5.53 (d, J=6.4 Hz, 1 H, OH-2'), 5.56 (d,
J=7.4 Hz, 1 H, H-1'), 5.71 (d, J=4.1 Hz, 1 H, OH-3'), 5.60 (d, J=7.4 Hz, 1
H, H-1'), 7.3 (m, 3 H), 8.42 (s, 1 H, H-8), 8.56 (br s, 1H, H--N.sup.6),
8.55 (br s, 1 H, NH--Me).
Example 50
This example describes the culture of Chinese hamster ovary (CHO) cells and
the preparation of a suspension of CHO cell membranes stably transfected
with rat A.sub.3 cDNA. These materials were used for the subsequent
experimental work set out herein.
CHO cells were transfected with rat A.sub.3 cDNA (Meyerhof et al.) using
methods well known to those of skill in the art. CHO cells stably
expressing the A.sub.3 receptor (Zhou et al.) were grown in F-12 (Ham's)
medium (Gibco BRL, Gaithersburg, Md.) containing 10% fetal bovine serum
(FBS, Gibco BRL) and penicillin/streptomycin (100 U/ml and 100 .mu.g/ml,
respectively; Gibco BRL) at 37.degree. C. in a 5% CO.sub.2 atmosphere.
When the transfected CHO cells had reached confluency, they were washed
twice with Dulbecco's phosphate buffer solution before dislodging after
addition of 3 ml trypsin-EDTA. For the final passage, cells were grown in
150.times.50 mm tissue culture dishes. Cells were washed twice with 10 ml
of ice-cold lysis buffer (10 mM Tris, 5 mM EDTA, pH 7.4, 5.degree. C.).
After addition of 5 ml of lysis buffer, cells were mechanically scraped
and homogenized in an ice-cold Dounce homogenizer (20 strokes by hand).
The suspension was centrifuged at 43,000.times.g for 10 min. The pellet
was resuspended in the minimum volume of ice-cold 50/10/1 buffer (50 mM
Tris, 10 mM MgCl.sub.2, 1 mM EDTA, pH 8.26, 5.degree. C.) required for the
binding assay and homogenized in a Dounce homogenizer. Typically, 6-8 175
cm.sup.2 flasks were used for a 48-tube assay. Adenosine deaminase (ADA,
Boehringer Mannheim, Indianapolis, Ind.) was added to a final
concentration of 3 U/ml, and the suspension was incubated at 37.degree. C.
for 15 min. The membrane suspension was subsequently kept on ice until
use. When large batches (ca 100 flasks) were processed, homogenization was
performed with a Polytron (Brinkman, Luzern, Switzerland) and further
work-up was as described above. The preparation was stored at -70.degree.
C. and retained its ›.sup.125 I!N.sup.6 -2-(4-aminophenyl)ethyladenosine
(›.sup.125 I!APNEA, prepared as described in Stiles et al., J. Biol.
Chem., 260, 10806-10811 (1985)) binding properties for at least one month.
Rat cerebral cortical and striatal membranes were prepared (Jacobson et
al., J. Med. Chem., 35, 4143-4149 (1992)) and treated with ADA for 30 min
at 37.degree. C. prior to storage at -70.degree. C.
Example 51
This example describes a radioligand binding assay used to study the
structure activity relationship (SAR) at the A.sub.3 receptor.
Binding of ›.sup.125 I!APNEA to CHO cells stably transfected with the
A.sub.3 receptor clone was performed essentially as described in Stiles et
al., J. Biol. Chem., 260, 10806-10811 (1985). Assays were performed in
50/10/1 buffer in glass tubes which contained 100 .mu.l of the membrane
suspension, 50 .mu.l of ›.sup.125 I!APNEA (final concentration 0.5 nM) or
›.sup.125 I!AB-MECA and 50 .mu.l of inhibitor. Inhibitors were routinely
dissolved in dimethylsulfoxide (DMSO) and were then diluted with buffer.
The final DMSO concentrations never exceeded 1%; this concentration did
not influence ›.sup.125 I!APNEA binding. Incubations were carried out in
duplicate for 1 hour at 37.degree. C., and were terminated by rapid
filtration over Whatman GF/B filters, using a Brandell cell harvester
(Brandell, Gaithersburg, Md.). Tubes were washed three times with 3 ml of
buffer. Radioactivity was determined in a Beckman gamma 5500B
.gamma.-counter. Non-specific binding was determined in the presence of 40
.mu.M N.sup.6 -›(R)-1-methyl-2-phenylethyl!adenosine (R-PIA). K.sub.i
-values were calculated according to Cheng-Prusoff (Cheng et al., Biochem.
Pharmacol., 22, 3099-3108 (1973)), assuming a K.sub.d for ›.sup.125
I!APNEA of 17 nM (Zhou et al.).
Binding of ›.sup.3 H!PIA (Amersham, Arlington Heights, Ill.) to A.sub.1
receptors from rat brain membranes and of ›.sup.3 H!CGS 21680 (DuPont NEN,
Boston, Mass.) to A.sub.2 receptors from rat striatal membranes was
performed as described previously (Jacobson et al. (1992)).
Solid samples of the adenosine derivatives were dissolved in DMSO and
stored in the dark at -20.degree. C. The stock solutions were diluted with
DMSO to a concentration of .ltoreq.0.1 mM prior to addition to the aqueous
medium. The final concentration of DMSO in the assay medium was generally
2%.
Binding data for a variety of adenosine derivatives are set forth in Table
3 below. At least six different concentrations spanning three orders of
magnitude, adjusted appropriately for the IC.sub.50 of each compound, were
used. IC.sub.50 values, computer-generated using a nonlinear regression
formula of the GraphPAD program (Institute of Scientific Information),
were converted to apparent K.sub.i values using K.sub.D values (Jacobson
et al. (1992)) of 1.0 and 14 nM for ›.sup.3 H!PIA and ›.sup.3 H!CGS 21680
binding, respectively, and the Cheng-Prusoff equation (Cheng et al.,
Biochem. Pharmacol., 22, 3099-3108 (1973)).
It can be seen that the preferred compounds of the present invention listed
in Table 3 have high binding affinities towards A3 receptors, and many of
the compounds of the instant invention also have selectivity for A3
receptors over receptors.
Effects on adenylate cyclase in CHO cells stably transfected with rat A3
adenosine receptors are given in Table 4.
FIG. 12 shows the inhibition of adenylate cyclase via rat A3 receptors in
transfected CHO cells: circles, NECA; squares, 2-Cl-IB-MECA; triangles,
compound 41b.
FIG. 13 shows the inhibition of adenylate cyclase via rat A3 receptors in
transfected CHO cells: circles, 2-chloro-N.sup.6
-(3-iodobenzyl)-adenosine-5'-N-methyluronamide; squares, NECA; triangles,
N6-benzyl-4'methyladenosine-5'-N-methyluronamide.
TABLE 1
__________________________________________________________________________
Characterization of 9-alkyl-adenine and ribose
modified adenosine derivatives.
Compound
No. m.p. (.degree.C.)
MS Formula Analysis
__________________________________________________________________________
3 159-161
366 (Cl)
C.sub.13 H.sub.12 N.sub.5 I.sub.1 0.3EtOAc
C, H, N
6 192-193
400 (Cl)
C.sub.13 H.sub.11 N.sub.5 Cl.sub.1 I.sub.1
C, H, N
9 203-205
381 (Cl)
C.sub.13 H.sub.13 N.sub.6 I.sub.1
.sup.a
10 202-203
396 (Cl)
C.sub.13 H.sub.14 IN.sub.7 I.sub.1.0.2C.sub.6 H.sub.14
C, H, N
11 185-186
395 (Cl)
C.sub.14 H.sub.15 N.sub.6 O.sub.1
.sup.a
12 190-191
409 (Cl)
C.sub.15 H.sub.17 N.sub.6 I.sub.1.0.6MeOH
C, H, N
13 134-135
423 (Cl)
C.sub.16 H.sub.19 N.sub.6 I.sub.1
C, H, N
14 138 465 (Cl)
C.sub.19 H.sub.25 N.sub.6 I.sub.1.0.35C.sub.6 H.sub.14
C, H, N
15 159 396 (Cl)
C.sub.14 H.sub.14 N.sub.5 O.sub.1 I.sub.1.0.2C.sub.6
H.sub.14.0.5MeOH
C, H, N
16 160-161
412 (Cl)
C.sub.14 H.sub.13 N.sub.5 S.sub.1 I.sub.1.0.35C.sub.6
H.sub.14 C, H, N
17 199 (dec.)
474 (Cl)
C.sub.18 H.sub.15 N.sub.6 S.sub.1 I.sub.1
.sup.a
18 185-187 C.sub.14 H.sub.14 IN.sub.5 O
C, H, N
19 125-128 C.sub.15 H.sub.16 IN.sub.5 O.sub.2.1H.sub.2 O
C, H, N
19b 130 C.sub.16 H.sub.18 IN.sub.5 O.sub.2 S.sub.1
.sup.a
20 126-127 C.sub.15 H.sub.16 IN.sub.5 O.sub.2
C, H, N
21 160 (d) C.sub.14 H.sub.12 IN.sub.5 O.sub.2.0.5H.sub.2 O
C, H, N
22 oil 418 (El)
C.sub.16 H.sub.15 IN.sub.6.1.5H.sub.2 O
.sup. C, H, N.sup.b
28 145-147 C.sub.16 H.sub.15 IN.sub.5 O.sub.3 Cl.sub.1 I.sub.1
C, H, N
29 158-161 C.sub.17 H.sub.19 N.sub.6 O.sub.3 I.sub.1
.sup.a
30 180-182
456 (Cl)
C.sub.16 H.sub.15 N.sub.5 O.sub.1 Cl.sub.1 I.sub.1
.sup.a
37 foam 571 (Cl)
C.sub.20 H.sub.20 N.sub.6 O.sub.4 Cl.sub.1 I.sub.1
.sup.a
40 130 387 (Cl)
C.sub.18 H.sub.19 N.sub.6 O.sub.2 Cl.sub.1
.sup.a
41 foam 607 (Cl)
C.sub.19 H.sub.20 N.sub.6 O.sub.5 Cl.sub.1 I.sub.1
S.sub.1 C, H, N
41b 162 528 (El)
C.sub.18 H.sub.18 N.sub.6 O.sub.3 Cl.sub.1 I.sub.1
C, H, N
42 184 553 (El)
C.sub.18 H.sub.17 N.sub.9 O.sub.2 Cl.sub.1 I.sub.1
.sup.a
43 98 528 (Cl)
C.sub.18 H.sub.19 N.sub.7 O.sub.2 Cl.sub.1 I.sub.1
.sup.a
44 119-129 C.sub.18 H.sub.17 N.sub.6 O.sub.2 Cl.sub.1 I.sub.1
F.sub.1.2H.sub.2 O
C, H, N
45 foam 759 (El)
C.sub.29 H.sub.43 N.sub.5 O.sub.5 Cl.sub.1 I.sub.1
Si.sub.1 .sup.a
46 120 (d) C.sub.19 H.sub.16 N.sub.6 O.sub.4 Cl.sub.1 I.sub.1
S.sub.1 C, H, N
__________________________________________________________________________
.sup.a High resolution mass in FAB+ mode m/z determined to be within
acceptable limits.
9: Calculated 381.0325. Found: 381.0335.
11: Calculated 395.0481. Found: 395.0463.
17: Calculated 475.0202. Found: 475.0201.
30: Calculated 456.0078. Found: 456.0077.
40: Calculated 386.1258. Found: 386.1249.
42: Calculated 553.0239. Found: 553.0226.
43: Calculated 527.0333. Found: 527.0318.
37: Calculated 571.0358. Found: 571.0361.
45: Calculated 760.1615. Found: 760.1614.
.sup.b 22, calc. 18.87% N; found 17.65%.
TABLE 2
__________________________________________________________________________
Elemental Analysis of 9-alkyl-adenosine and
ribose-modified adenosine derivatives.
Compound
No. Formula Calculated
Found
__________________________________________________________________________
3 C.sub.13 H.sub.12 N.sub.5 I.sub.1.0.3EtOAc
C, 43.55; H, 3.71;
C, 43.66; H, 3.86;
N, 17.88 N, 18.02
6 C.sub.13 H.sub.11 N.sub.5 Cl.sub.1 I.sub.1
C, 39.07; H; 2.77;
C, 39.35; H, 2.74;
N, 17.52 N, 17.55
10 C.sub.13 H.sub.14 N.sub.7 I.sub.1.0.2C.sub.6 H.sub.14
C, 41.35; H, 4.10;
C, 41.33; H, 3.88;
N, 23.77 N, 23.88
12 C.sub.15 H.sub.17 N.sub.6 I.sub.1.0.6MeOH
C, 43.83; H, 4.57;
C, 43.70; H, 4.17;
N, 19.66 N, 19.28
13 C.sub.16 H.sub.19 N.sub.6 I.sub.1
C, 45.51; H, 4.54;
C, 45.74; H, 4.59;
N, 19.79 N, 19.79
14 C.sub.19 H.sub.25 N.sub.6 I.sub.1.0.35C.sub.6 H.sub.14
C, 51.25; H, 6.09;
C, 51.24; H, 5.85;
N, 16.99 N, 17.07
15 C.sub.14 H.sub.14 N.sub.5 O.sub.1 I.sub.1.0.2C.sub.6 H.sub.14.0.5Me
OH C, 44.03; H, 4.42;
C, 44.04; H, 4.24;
N, 16.35 N, 16.01
16 C.sub.14 H.sub.13 N.sub.5 O.sub.3.0.35C.sub.6 H.sub.14
C, 43.90; H, 4.10;
C, 43.97; H, 4.02;
N, 15.90 N, 16.67
18 C.sub.14 H.sub.14 IN.sub.5 O
C, 42.54; H, 3.57;
C. 42.51; H, 3.60;
N, 17.72 N, 17.75
19 C.sub.15 H.sub.16 IN.sub.5 O.sub.2.1H.sub.2 O
C, 40.64; H, 4.09;
C, 40.67; H, 4.14;
N, 15.80 N, 15.40
20 C.sub.15 H.sub.16 IN.sub.5 O.sub.2
C, 42.36; H, 3.79;
C, 42.31; H, 3.97;
N, 15.74 N, 15.74
21 C.sub.14 H.sub.12 IN.sub.5 O.sub.2.0.5H.sub.2 O
C, 40.31; H, 3.13;
C, 40.35; H, 3.24;
N, 16.74 N, 16.76
22 C.sub.16 H.sub.15 IN.sub.6.1.5H.sub.2 O
C, 43.16; H, 4.08;
C, 43.10; H, 3.70;
N, 18.87 N, 17.65
28 C.sub.19 H.sub.15 N.sub.5 O.sub.3 Cl.sub.1 I
C, 39.40; H, 3.10;
C, 38.48; H, 3.06;
N, 14.36 N, 14.72
41b C.sub.18 H.sub.18 N.sub.6 O.sub.3 Cl.sub.1 I
C, 43.79; H, 4.49;
C, 43.81; H, 4.14;
N, 14.18 N, 14.81
41 C.sub.19 H.sub.20 N.sub.6 O.sub.5 Cl.sub.1 S.sub.1 I.sub.1
C, 37.61; H, 3.32;
C, 37.64; H, 3.37;
N, 13.85 N, 13.75
44 C.sub.18 H.sub.17 N.sub.6 O.sub.2 Cl.sub.1 I.sub.1 F.sub.1.2H.sub.2
O C, 38.14; H, 3.74;
C, 38.53; H, 4.06;
N, 14.82 N, 14.68
46 C.sub.19 H.sub.16 N.sub.6 O.sub.4 Cl.sub.1 I.sub.1 S.sub.1
C, 38.89; H, 2.75;
C, 38.63; H, 2.90;
N, 14.32 N, 14.28
__________________________________________________________________________
TABLE 3
__________________________________________________________________________
Affinities of 9-alkyl adenine and ribose-modified adenosine derivatives
in
radioligand bindinq assays at rat brain A.sub.1, A.sub.2a, and A.sub.3
receptors..sup.a-c
K.sub.i (.mu.M) or % Inhibition
Cmpd.
No. Compound Name K.sub.i (A.sub.1).sup.a
K.sub.i (A.sub.2a).sup.b
K.sub.i (A.sub.3).sup.c
A.sub.i /A.sub.3
A.sub.2a /A.sub.3
__________________________________________________________________________
3 N.sup.6 -(3-Iodobenzyl)-9-
5.73 .+-. 1.88
2.23 .+-. 1.33
48.3 .+-. 6.0
0.12
0.046
methyladenine
6 2-Chloro-N.sup.6 -(3-iodobenzyl)-
0.45 .+-. 0.11
2.7 .+-. 0.56
51.0 .+-. 10.0
0.0088
0.053
9-methyladenine
9 2-Amino-N.sup.6 -(3-iodobenzyl)-9-
5.57 .+-. 1.32
3.22 .+-. 1.52
40.1 .+-. 5.7
0.14
0.080
methyladenine
10 2-Hydrazido-N.sup.6 -(3-iodobenzyl)-
5.44 .+-. 0.05
19.6 .+-. 7.8
109 .+-. 9
0.050
0.18
9-methyladenine
11 N.sup.6 -(3-Iodobenzyl)-2-
0.648 .+-. 0.102
3.56 .+-. 0.84
0.974 .+-. 0.340
0.67
3.7
methylamino-9-methyladenine
12 2-Dimethylamino-N.sup.6 -(3-
1.48 .+-. 0.12
9.89 .+-. 3.01
15.0 .+-. 0.9
0.099
0.66
iodobenzyl)-9-methyladenine
13 N.sup.6 -(3-Iodobenzy1)-9-methyl-2-
0.33 .+-. 0.08
1.72 .+-. 0.70
20%(30 .mu.M)
<<1 <<1
propylaminoadenine
14 2-Hexylamino-N.sup.6 -(3-iodo
4.48 .+-. 0.82
11 .+-. 4%
19%(30 .mu.M)
<1 --
benzyl)-9-methyladenine (10.sup.-5)
15 N.sup.6 -(3-Iodobenzyl)-2-methoxy-9-
0.50 .+-. 0.21
1.24 .+-. 0.11
18.3 .+-. 12.9
0.027
0.068
methyladenine
16 N.sup.6 -(3-Iodobenzyl)-9-methyl-2-
1.89 .+-. 0.59
1.64 .+-. 0.39
0.299 .+-. 0.074
6.3 5.5
methylthioadenine
17 N.sup.6 -(3-Iodobenzyl)-9-methyl-2-
0.84 .+-. 0.19
11.6 .+-. 4.0
166 .+-. 57
0.0051
0.070
(4-pyridylthio)adenine
18 N6-(3-Iodobenzyl)-9-
22.9 .+-. 3.7
15.1 .+-. 1.6
62.5 .+-. 14.5
0.37
0.24
hydroxyethyladenine
19 R-N.sup.6 -(3-Iodobenzyl)-9-(2,3-
13.8 .+-. 2.2
18.9 .+-. 1.9
24.9 .+-. 10.7
0.55
0.76
dihydroxypropyl)adenine
19b R-N.sup.6 -(3-Iodobenzyl)-
1.34 .+-. 0.09
78.9 .+-. 23.5
8.59 .+-. 4.29
0.16
9.2
9-(2,3-dihydroxypropyl)-
2-methylthioadenine
20 S-N.sup.6 -(3-Iodobenzyl)-9-(2,3-
19.1 .+-. 2.2
41.8 .+-. 12.5
142 .+-. 13
0.13
0.29
dihydroxypropyl)adenine
21 N.sup.6 -(3-Iodobenzyladenin-9-yl)acetic
17% (10.sup.-4)
9% (10.sup.-4)
225 .+-. 17
-- --
acid
22 N.sup.6 -(3-iodobenzyl)-9-(3-
6.03 .+-. 1.37
18.7 .+-. 5.8
185 .+-. 17
0.032
0.10
cyanopropyl)adenine
28 2-Chloro-9-(.beta.-
0.811 .+-. 0.123
2.89 .+-. 1.00
0.276 .+-. 0.110
2.9 10
D-erythrofuranoside)-
N.sup.6 -(3-iodobenzyl) adenine
29 9-(.beta.-D-Erythrofuranoside)-2-
0.660 .+-. 0.010
3.39 .+-. 0.29
69 0.0096
0.049
methylamino-N.sup.6 -(3-iodobenzyl)
adenine
30 2-Chloro-N-(3-iodobenzyl)-9-
0.174 .+-. 0.017
4.12 .+-. 0.18
3.47 .+-. 0.58
0.13
>>1
(2-tetrahydrofuryl)-9H-purin-6-amine
37 9-(2-Acetyl-3-deoxy-.beta.-D-5-
0.778 .+-. 0.044
15.9 .+-. 3.7
0.0625 .+-. 0.0310
12 250
methyl-ribofuronamido)-2-chloro-
N.sup.6 -(3-iodobenzyl)adenine
40 2-Chloro-9-(2,3-dideoxy-.beta.-D-5-methyl
11.5 .+-. 1.3
220 .+-. 65
30.9 .+-. 1.3
0.37
7.1
ribofuronamido)-N.sup.6 - benzyladenine
41 2-Chloro-9-(3-deoxy-2-methane
1.29 .+-. 0.08
41.9 .+-. 6.2
7.27 .+-. 1.19
0.18
5.8
sulfonyl-.beta.D-5-methyl-
ribofuronamido)-N.sup.6 -(3-iodobenzyl)adenine
41b 2-Chloro-9-(3-deoxy-.beta.-D-5-
1.03 .+-. 0.15
4.66 .+-. 0.74
0.0329 .+-. 0.0078
31 140
methylribofuronamido)-N.sup.6 -(3-
iodobenzyl)adenine
42 2-Chloro-9-(2'-azido-2',3'-dideoxy-
0.401 .+-. 0.041
28.1 .+-. 3.2
6.01 .+-. 0.63
0.067
4.7
.beta.-D-5'-methyl-arabino- furonamido)
N.sup.6 -benzyladenine
43 2-Chloro-9-(2'-amino-2',3'-dideoxy-
6.69 .+-. 0.74
2% (10.sup.-4)
3.40 .+-. 0.79
2.0 >50
.beta.-D-5'-methyl-arabino
furonamido)-N.sup.6 -(3-iodobenzyl)adenine
44 2-Chloro-9-(2',3'-dideoxy-2'-fluoro-
1.42 .+-. 0.27
98.0 .+-. 9.7
17.8 .+-. 2.4
0.080
5.5
.beta.-D-5'-methylarabinofuronamido)-
N.sup.6 -(3-iodobenzyl)adenine
45 2-Chloro-9-(3,5-1,1,3,3-tetra-
66.3 .+-. 19.8
18 .+-. 2%
13.1 .+-. 3.5
5.1 >7
isopropyldisiloxyl-.beta.-D-5-
(10.sup.-4)
ribofuranosyl)-
N.sup.6 -(3-iodobenzyl)adenine
46 2-Chloro-9-(2',3'-O-
0.179 .+-. 0.024
0.871 .+-. 0.219
0.0122 .+-. 0.0013
15 71
thiocarbonyl-.beta.-D-5-methyl-
ribofuronamido)-
N.sup.6 -(3-iodobenzyl)adenine
51 (1S, 2R, 3S, 4R)-4-(6-amino-2-
0.946 .+-. 0.179
1.82 .+-. 0.20
59.2 .+-. 9.2
--
phenylethylamino-9H-purin-9-
yl)cyclopentane-1,2,3-triol
56 1-(6-benzylamino-9H-purin-9-yl)-1-
62.4 .+-. 6.1
53.6 .+-. 14.6
0.604 .+-. .143
deoxy-N,4-dimethyl-.beta.-D-
ribofuranosiduronamide
61 (.+-.)-9-›2.alpha.,3.alpha.-Dihydroxy-4.beta.-(N-
35.9 .+-. 8.3
28 .+-. 5%
19.5 .+-. 4.7
1.8 >1
methylcarbamoyl) (10.sup.-4)
cyclopent-1.beta.-yl)!-N.sup.6 -(3-
iodobenzyl)adenine
65 N.sup.6 -›3-(L- 170 .+-. 30
215 .+-. 54
pKi 0.014
0.018
prolylamino)benzyl!adenosine-5'-N-
4.93
methyluronamide
66 N.sup.6 -›3(.beta.-
101 .+-. 9
144 .+-. 40
pKi 4.5 6.3
alanylamino)benzyl!adenosine-5'- 7.64
methyluronamide
67 N.sup.6 -›3-(N-6-Boc-.beta.-
4,500 .+-. 1,050
1,960 .+-. 410
pKi 96 42
alanylamino)benzyl!adenosine-5'- 7.33
methyluronamide
70 6-(N'-phenylhydrazinyl)purine-9-.beta.-
3,940 .+-. 240
7,160 .+-. 80
pKi 18 31
ribofuranoside-5'-N-methyluronamide
6.65
71 6-O-phenyl hydroxylamino)purine-9-.beta.-
2,060 .+-. 370
66,300 .+-. 16,200
pKi 50 2
ribofuranoside-5'-N-methyluronamide
5.87
74 1-(6-Furfurylamino-9H-purin-9-yl)-1-
8.61 .+-. 2.55
6.22 .+-. 2.71
0.720 .+-. 250
deoxy-N-methyl-.beta.-D-
ribofuranosiduronamide
2-Chloro-9-(2',3'-dibenzoyl-.beta.-D-5-
21% (10.sup.-4)
7% (10.sup.-4)
55 .+-. 2% (10.sup.-4)
-- --
methyl-ribofuronamido)-N.sup.6 -
(3-iodobenzyl)adenine
9-(.beta.-talosyl)adenine
150 .+-. 28
54.7 .+-. 3.1
6% (10.sup.-4)
<1 <1
2-Chloro-9-(.beta.-arabinosyl)
24.2 .+-. 7.9
90.0 .+-. 12.7
14% (10.sup.-5)
-- --
adenine
__________________________________________________________________________
.sup.a Displacement of specific ›.sup.3 H!PIA binding, unless noted, in
rat brain membranes expressed as Ki .+-. S.E.M. in .mu.M (n = 3-6).
.sup.b Displacement of specific ›.sup.3 H!CGS 21680 binding, unless noted
in rat brain membranes expressed as Ki .+-. S.E.M. in .mu.M (n = 3-6).
.sup.c Displacement of specific binding of ›.sup.125
I!N.sup.6(4-amino-3-iodobenzyl)adenosine-5N-methyluronamide from membrane
of CHO cells stably transfected with the rat A.sub.3cDNA, expressed as Ki
.+-. S.E.M. in .mu.M (n = 3-7).
TABLE 4
______________________________________
Effects on adenylate cyclase in CHO cells
stably transfected with rat A.sub.3 adenosine
receptors..sup.a
Compound
Conc. Ratio Conc./ Effect on IB-MECA
No. (.mu.M)
K.sub.i (A3)
% Inhib..sup.b
dose-resp. curve
______________________________________
16 100 330 19.5 c
19b 40 4.6 7.4 .+-. 3.7
c
28 100 360 42.2 n.d.
37 20 320 12.7 .+-. 1.0
n.d.
40 40 1.3 18.1 .+-. 8.7
c
41b 100 3000 49.2 .+-. 3.7
n.d.
42 100 17 27.8 n.d.
44 100 5.6 11.2 n.d.
45 100 7.6 8.7 n.d.
46 100 8200 54.5 .+-. 8.1
n.d.
______________________________________
.sup.a in the presence of 1 .mu.M forskolin.
.sup.b average .+-. S.E.M. for 2-3 determinations or a single value.
.sup.c no effect
n.d. not determined
All of the references, including patents, patent applications, and
publications, cited herein are hereby incorporated in their entireties by
reference.
While this invention has been described with an emphasis upon the preferred
embodiment, it will be obvious to those of ordinary skill in the art that
variations of the preferred embodiment may be used and that it is intended
that the invention may be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all modifications
encompassed within the spirit and scope of the invention as defined by the
following claims.
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